Combination therapy for ischemia

ABSTRACT

The invention provides a combination treatment for ischemia conditions in or otherwise affecting the CNS, such as stroke. The treatment involves administration of a PSD-95 inhibitor and performing reperfusion therapy (e.g., by administration of tPA). Administering a PSD-95 inhibitor in combination with reperfusion therapy increases the efficacy of the reperfusion therapy and/or slows the decline in efficacy of reperfusion therapy with time after onset of ischemia thus extending the window in which reperfusion therapy can be administered.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is continuation of U.S. Ser. No. 14/128,941 filed May21, 2014, which is the national stage of PCT/IB2012/053178 filed Jun.23, 2012, which is a non-provisional and claims the benefit of U.S.61/501,117, filed Jun. 24, 2011, and U.S. 61/617,001, filed Mar. 28,2012, all of which are incorporated by reference in their entirety forall purposes.

REFERENCE TO A SEQUENCE LISTING

The sequence listing in file 514905_SEQLIST.TXT was created Jul. 31,2018, and is 17,501 bytes. This sequence listing is hereby incorporatedby reference.

BACKGROUND

Ischemic stroke is a common cause of death and serious disability and isusually caused by a blockage in a blood vessel leading to or within theintracranial cavity and/or brain. Few effective treatments areavailable. One treatment consists of removing the blockage within theblood vessel in question. Other treatments consist of altering perfusionpressures within the brain by increasing blood pressure to the brain.Blockage of blood vessels can be removed using a range of mechanicaldevices, or using “clot busting agents” which are deliveredintravenously or intra-arterially. Among such clot busting agents isTissue plasminogen factor (tPA), a thrombolytic agent that isadministered to some stroke subjects to dissolve emboli causing theischemia and thus restore blood flow to the brain, and recombinant tPA'ssuch as Alteplase, reteplase and tenecteplase. Other thrombolytic drugsthat break down clots include streptokinase, urokinase and desmotaplase.Among mechanical reperfusion devices, there are intra-arterialcatheters, balloons, stents, and various clot retrieval devices, such asthe Penumbra System Reperfusion Cather. Among treatments that alterperfusion pressures in the brain are devices that increase the arterialpressure in the brain, such as balloons that can be inflated in theextra-cerebral arteries such as the aorta thereby diverting blood flowfrom other body areas and increasing brain arterial perfusion, such asthe CoAxia NeuroFlo™ catheter device. Collectively, these strategies canbe considered as medical and mechanical agents that enhance brainperfusion on or after the onset of cerebral ischemia (hereaftercollectively “reperfusion therapies”).

Although tPA and other reperfusion therapies administered soon afteronset of ischemia are effective in reducing death or disability fromischemic stroke, less than about 3% of subjects presenting with strokeare treated with tPA or other reperfusion therapies. The low usage oftPA and other reperfusion therapies is due in part to the risk of deathif administered to a patient who is having or who is at an elevated riskfor sustaining a brain hemorrhage. Stroke can be the result of ischemiaor hemorrhage. Too often, the time required to bring a subject to ahospital, reach an initial diagnosis and perform a brain scan todistinguish between ischemic and hemorrhagic stroke would place asubject outside the window in which tPA or other reperfusion therapiescan be effective. Thus, many ischemic stroke subjects, who could benefitfrom tPA or other reperfusion therapies, do not receive such treatment.

A different form of treatment for stroke and related conditions is nowin clinical trials (see WO 2010144721 and Aarts et al., Science 298,846-850 (2002)). This treatment uses Tat-NR2B9c (NA-1), an agent thatinhibits PSD-95, thus disrupting binding to N-methyl-D-aspartatereceptors (NMDARs) and neuronal nitric oxide synthases (nNOS) andreducing excitoxicity induced by cerebral ischemia. Treatment reducesinfarction size and functional deficits.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, B, C, and D: Description of the protocol for dosing non-humanprimates (NHPs; A), and graphs of the resulting diffusion volumes on MRIindicating areas of damage. B. DWI MRI 48 hours after the onset of a 4.5hour stroke. C. T2 volume 48 house after the onset of a 4.5 hour stroke.D. T2 volume 7 days after the onset of a 4.5 hour stroke.

FIGS. 2A, B. A. Animals were subjected to 4.5 hour MCAO and treatedwithin 5 min with Tat-NR2B9c or placebo. Time course of increase in DWIhyperintensity after MCAO in treated and control animals. B. Perfusionand MRI images of brain at different time points.

FIG. 3. Temporal evolution of penumbra mismatch in placebo or Tat-NR2B9canimals.

FIGS. 4A, B. Tat-NR2B9c reduces intracellular ATP depletion and protectsmouse cortical neurons against cytotoxicity induced by oxygen-glucosedeprivation (OGD). (A) Fraction of cell death as measured 20 hours afterOGD by propidium iodide labeling method. (B) Intracellular ATPconcentration from cortical neurons determined by a chemiluminescent ATPdetection assay, expressed as % ATP concentration relative to normoxiccontrol samples.

FIG. 5: Demonstration that NA-1 (Tat-NR2B9c), when administered as asingle dose after a stroke, can halt the development of lesions in thebrain as assessed by Magnetic Resonance Imaging (MRI). This efficacydoes not act through the modification of cerebral blood flow.

FIG. 6A-F: A: Volumes of perfusion defects at baseline. B: Analysis ofstroke volumes as measured by DWI and T2 imaging over 30 days. C:Representative T2-weighted images of strokes incurred in placebo anddrug treated animals 24 hr after MCAO. D: Representative serialhistological sections from NA-1 (Tat-NR2B9c) and placebo treated animalsat 30 days stained with haematoxylin and eosin. E: Stroke volumescalculated using 24 hr DWI volumes and 30-day T2-weighted volumes. F:NHPSS over the 30 day observation period.

FIGS. 7A-F: A: volume of perfusion defects. B: Stroke volumes as measureby DWI and T2 MRI over 7 days. C: Stroke volumes from 48 hr DWI and T2-and 7-day T2-weighted MRI scans normalized to each animal's initialperfusion deficit. D: Representative 7-day MRI. E: Representative 7-dayhistology. F: NHPSS scores over the 7-day observation period.

FIGS. 8A-F: A: volume of perfusion defects. B: Stroke volumes as measureby DWI and T2 MRI over 14 days. C: Stroke volumes from 48 hr DWI and T2-and 14-day T2-weighted MRI scans normalized to each animal's initialperfusion deficit. D: Representative 14-day MRI. E: Representative14-day histology. F: NHPSS scores over the 14-day observation period.

FIGS. 9A, B:A: Graph showing the ratio of PSD-95:NMDARco-immunoprecipitation between the ipsilateral and contralateralhemispheres of rats following a stroke and treatment with NA-1(Tat-NR2B9c). B: Example immunoblots showing the amount of NMDARimmunoprecipitated with an anti-PSD-95 antibody in the presence ofvarious concentrations of NA-1 or controls.

FIG. 10: Graph showing the ratio of PSD-95:NMDAR co-immunoprecipitationbetween the ipsilateral and contralateral hemispheres of rats atdifferent timepoints following a stroke and treatment with NA-1(Tat-NR2B9c).

FIG. 11: Infarct areas in rat brains 24 hours after being subjected to astroke and treated with various combinations and times of tPA and NA-1(Tat-NR2B9c) dosing.

SUMMARY OF THE CLAIMED INVENTION

The invention provides a method of treating a damaging effect ofischemia on the central nervous system, comprising administering aPSD-95 inhibitor to a subject having or at risk of ischemia, andperforming reperfusion therapy on the subject, wherein thePSD95-inhibitor and reperfusion therapy treat a damaging effect of theischemia on the central nervous system of the subject. Optionally, thePSD-95-inhibitor is administered before reperfusion therapy isperformed. Optionally, the PSD-95-inhibitor is administered to a subjectat risk of ischemia before onset of ischemia and the reperfusion therapyis performed after onset of ischemia. Optionally, the PSD-95-inhibitoris administered and reperfusion therapy is performed after onset ofischemia. In some methods, the ischemia is cerebral ischemia. In somemethods, the subject has a stroke. In some methods, the ischemia iscardiac, pulmonary or major limb ischemia affecting the central nervoussystem by inhibiting blood flow to or from the CNS. In some methods, thesubject is tested for presence of cerebral ischemia and/or absence ofcerebral hemorrhage between administration of the agent and performanceof the reperfusion therapy. In some methods, the subject is assessed forpresence or risk of hemorrhage between administering the agent andperformance of the reperfusion therapy. In some methods, the assessmentincludes performing a PET scan, CAT scan, MRI or reviewing the subject'smedical history or the use of one or more biomarkers providing anindication of ischemia. In some methods, the PSD-95-inhibitor is apeptide. In some methods, the agent is NA-1 (Tat-NR2B9c). In somemethods, the reperfusion is performed by administering a thrombolyticagent. In some methods, the thrombolytic agent is a plasminogenactivator. In some methods, the thrombolytic agent is tPA. In somemethods, the reperfusion therapy is mechanical reperfusion. In somemethods, the reperfusion therapy is performed more than 3 hours afteronset of ischemia. In some methods, the reperfusion therapy is performedmore than 4.5 hours after onset of ischemia. In some methods, thereperfusion therapy is performed more than 4.5 hours and less than 24hours after onset of ischemia. In some methods, the reperfusion therapyis performed after determining the subject qualifies for reperfusionbased on lack of a completed infarction, an ischemic penumbra and lackof hemorrhage as shown by CT, MRI or PET analysis. In some methods, thereperfusion therapy is performed at least 12 or at least 24 hours afteronset of ischemia. In some methods, the reperfusion therapy is performed275-690 minutes after onset of ischemia. In some methods, the intervalbetween administering PSD-95 and reperfusion therapy can be 30 min to 6hr. In some methods, a thrombolytic agent is administered by localizedadministration to a site of impaired blood flow. In any of the abovemethods, the peptide or other agent can be linked to an internalizationpeptide or lipidated thereby facilitating passage of the peptide acrossa cell membrane or the blood brain barrier. Some peptides or otheragents are myristoylated. Peptides are preferably myristoylated at theN-terminus.

The invention further provides a method of treating a subject populationpresenting sign(s) and/or symptom(s) of ischemia, comprisingadministering a PSD-95 inhibitor to the subjects; wherein the subjectsare analyzed for unacceptable risk of side effects of reperfusiontherapy, and subjects without unacceptable risk of side effects receivereperfusion therapy and subjects with unacceptable risk of side effectsdo not receive reperfusion therapy. In some methods, the analysis ofunacceptable risk of side effects includes analysis for presence or riskof hemorrhage. In some methods, the subjects present sign(s) and/orsymptom(s) of stroke and the analysis includes performing a brain scanthat distinguishes ischemic stroke and hemorrhagic stroke and subjectshaving ischemic stroke receive the reperfusion therapy and subjectshaving hemorrhagic stroke do not.

The invention provides an agent that inhibits PSD-95 binding to NMDAR 2Bor other NMDAR 2 subunit(s) for use in treating a damaging effect ofischemia on the central nervous system in a subject also receivingreperfusion therapy, wherein the reperfusion therapy and agent treatdamaging effects of the ischemia on the central nervous system.

The invention further provides an agent or device for use in reperfusiontherapy in a subject also receiving an agent that inhibits PSD-95binding to NMDAR 2B, 2A or nNOS wherein the reperfusion therapy and theagent treat a damaging effect of the ischemia on the central nervoussystem. Optionally, the device is a coil, stent, balloon (e.g., anintra-aortic balloon, pump), catheter. Optionally, the agent is athrombolytic, vasodilator or hypertensive agent.

Definitions

A “chimeric peptide” means a peptide having two component peptides notnaturally associated with one another joined to one another as a fusionprotein or by chemical linkage.

A “fusion” protein or polypeptide refers to a composite polypeptide,i.e., a single contiguous amino acid sequence, made up of sequences fromtwo (or more) distinct, heterologous polypeptides which are not normallyfused together in a single polypeptide sequence.

The term “PDZ domain” refers to a modular protein domain of about 90amino acids, characterized by significant sequence identity (e.g., atleast 60%) to the brain synaptic protein PSD-95, the Drosophila septatejunction protein Discs-Large (DLG), and the epithelial tight junctionprotein ZO1 (ZO1). PDZ domains are also known as Discs-Large homologyrepeats (“DHRs”) and GLGF (SEQ ID NO:7) repeats. PDZ domains generallyappear to maintain a core consensus sequence (Doyle, D. A., 1996, Cell85: 1067-76). Exemplary PDZ domain-containing proteins and PDZ domainsequences disclosed in U.S. application Ser. No. 10/714,537, which isherein incorporated by reference in its entirety.

The term “PL protein” or “PDZ Ligand protein” refers to a naturallyoccurring protein that forms a molecular complex with a PDZ-domain, orto a protein whose carboxy-terminus, when expressed separately from thefull length protein (e.g., as a peptide fragment of 3-25 residues, e.g.3, 4, 5, 8, 9, 10, 12, 14 or 16 residues), forms such a molecularcomplex. The molecular complex can be observed in vitro using the “Aassay” or “G assay” described, e.g., in US 20060148711, or in vivo.

The term “NMDA receptor,” or “NMDAR,” refers to a membrane associatedprotein that is known to interact with NMDA including the varioussubunit forms described below. Such receptors can be human or non-human(e.g., mouse, rat, rabbit, monkey).

A “PL motif” refers to the amino acid sequence of the C-terminus of a PLprotein (e.g., the C-terminal 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20 or25 contiguous residues) (“C-terminal PL sequence”) or to an internalsequence known to bind a PDZ domain (“internal PL sequence”).

A “PL peptide” is a peptide of comprising or consisting of, or otherwisebased on, a PL motif that specifically binds to a PDZ domain.

The terms “isolated” or “purified” means that the object species (e.g.,a peptide) has been purified from contaminants that are present in asample, such as a sample obtained from natural sources that contain theobject species. If an object species is isolated or purified it is thepredominant macromolecular (e.g., polypeptide) species present in asample (i.e., on a molar basis it is more abundant than any otherindividual species in the composition), and preferably the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, an isolated, purified orsubstantially pure composition comprises more than 80 to 90 percent ofall macromolecular species present in a composition. Most preferably,the object species is purified to essential homogeneity (i.e.,contaminant species cannot be detected in the composition byconventional detection methods), wherein the composition consistsessentially of a single macromolecular species. The term isolated orpurified does not necessarily exclude the presence of other componentsintended to act in combination with an isolated species. For example, aninternalization peptide can be described as isolated notwithstandingthat it is linked to an active peptide.

A “peptidomimetic” refers to a synthetic chemical compound which hassubstantially the same structural and/or functional characteristics of apeptide consisting of natural amino acids. The peptidomimetic cancontain entirely synthetic, non-natural analogues of amino acids, or canbe a chimeric molecule of partly natural peptide amino acids and partlynon-natural analogs of amino acids. The peptidomimetic can alsoincorporate any amount of natural amino acid conservative substitutionsas long as such substitutions also do not substantially alter themimetic's structure and/or inhibitory or binding activity. Polypeptidemimetic compositions can contain any combination of nonnaturalstructural components, which are typically from three structural groups:a) residue linkage groups other than the natural amide bond (“peptidebond”) linkages; b) non-natural residues in place of naturally occurringamino acid residues; or c) residues which induce secondary structuralmimicry, i.e., to induce or stabilize a secondary structure, e.g., abeta turn, gamma turn, beta sheet, alpha helix conformation, and thelike. In a peptidomimetic of a chimeric peptide comprising an activepeptide and an internalization peptide, either the active moiety or theinternalization moiety or both can be a peptidomimetic.

The term “specific binding” refers to binding between two molecules, forexample, a ligand and a receptor, characterized by the ability of amolecule (ligand) to associate with another specific molecule (receptor)even in the presence of many other diverse molecules, i.e., to showpreferential binding of one molecule for another in a heterogeneousmixture of molecules. Specific binding of a ligand to a receptor is alsoevidenced by reduced binding of a detectably labeled ligand to thereceptor in the presence of excess unlabeled ligand (i.e., a bindingcompetition assay).

Excitotoxicity is the pathological process by which neurons are damagedand killed by the overactivation of receptors for the excitatoryneurotransmitter glutamate, such as the NMDA receptors, e.g., NMDAreceptors bearing the NMDAR 2B subunit.

The term “subject” includes humans and veterinary animals, such asmammals, as well as laboratory animal models, such as mice or rats usedin preclinical studies.

The term “agent” includes any compound including compounds with orwithout pharmaceutical activity, natural compounds, synthetic compounds,small molecules, peptides and peptidomimetics. A PSD-95 inhibitor is anagent that inhibits PSD-95 as further described below.

The term “pharmacologic agent” means an agent having a pharmacologicalactivity. Pharmacological agents include compounds that are known drugs,compounds for which pharmacological activity has been identified butwhich are undergoing further therapeutic evaluation in animal models orclinical trials. A chimeric agent comprises a pharmacologic agent linkedto an internalization peptide. An agent can be described as havingpharmacological activity if it exhibits an activity in a screeningsystem that indicates that the active agent is or may be useful in theprophylaxis or treatment of a disease. The screening system can be invitro, cellular, animal or human. Agents can be described as havingpharmacological activity notwithstanding that further testing may berequired to establish actual prophylactic or therapeutic utility intreatment of a disease.

A tat peptide means a peptide comprising or consisting of GRKKRRQRRR(SEQ ID NO:1), in which no more than 5 residues are deleted, substitutedor inserted within the sequence, which retains the capacity tofacilitate uptake of a linked peptide or other agent into cells.Preferably any amino acid changes are conservative substitutions.Preferably, any substitutions, deletions or internal insertions in theaggregate leave the peptide with a net cationic charge, preferablysimilar to that of the above sequence. Such can be accomplished by notsubstituting or deleting a significant number of R and K residues. Theamino acids of a tat peptide can be derivatized with biotin or similarmolecule to reduce an inflammatoy response.

Co-administration of a pharmacological agents means that the agents areadministered sufficiently close in time for detectable amounts of theagents to present in the plasma simultaneously and/or the agents exert atreatment effect on the same episode of disease or the agents actco-operatively, or synergistically in treating the same episode ofdisease. For example, an anti-inflammatory agent acts cooperatively withan agent including a tat peptide when the two agents are administeredsufficiently proximately in time that the anti-inflammatory agent caninhibit an anti-inflammatory response inducible by the internalizationpeptide.

Statistically significant refers to a p-value that is <0.05, preferably<0.01 and most preferably <0.001.

An episode of a disease means a period when signs and/or symptoms of thedisease are present interspersed by flanked by longer periods in whichthe signs and/or symptoms or absent or present to a lesser extent.

DETAILED DESCRIPTION I. General

The present invention provides a combination treatment for ischemia inor otherwise affecting the CNS, such as ischemic stroke. The treatmentinvolves administration of a PSD95 inhibitor and performing areperfusion therapy (e.g., by administration of tPA or anotherthrombolytic agent, or by using a mechanical device to increase bloodflow to the affected CNS area). In conventional use of tPA and otherreperfusion therapies, the efficacy declines with increasing time fromonset of ischemia and the potential for hemorrhagic side effectsincreases. Thus, in the case of tPA, this thrombolytic strategy isconsidered ineffective after about 3-4.5 hr from onset of ischemia. Theinvention is based in part on the insight that administering a PSD95inhibitor in combination with a reperfusion therapy increases theefficacy of the reperfusion therapy and/or slows the decline in efficacyof tPA or other reperfusion therapies with time after onset of ischemiathus extending the window in which tPA or other reperfusion therapiescan be administered.

Whereas tPA and other reperfusion therapies can be safely administeredonly to stroke subjects known to have ischemic stroke, the PSD-95inhibitor can be administered safely to any stroke or possible strokesubject, irrespective whether the subject has ischemic or hemorrhagicstroke and irrespective whether the subject has suffered a stroke atall. By administering the PSD-95 inhibitor, there is more time availableto perform a brain scan or any other diagnostic test in order todetermine presence of ischemic stroke, and then administer tPA oranother reperfusion therapy if appropriate. Thus, more subjects withischemic stroke can benefit from tPA or other reperfusion therapies andat the same time benefit from treatment with a PSD-95 inhibitor.

II. Agents Inhibiting PSD-95

PSD-95 inhibitors inhibit interaction between PSD-95 and one or moreNMDARs (e.g., 2A, 2B, 2C or 2D) or nNOS (e.g., Swiss-Prot P29475).Inhibition can be, for example, the result of specific binding of theinhibitor to PSD-95. Such agents are useful for reducing one or moredamaging effects of stroke and other neurological conditions mediated atleast in part by NMDAR excitotoxicity. Such agents include peptideshaving an amino acid sequence including or based on the PL motif of aNMDA Receptor or PDZ domain of PSD-95. Such peptides can also inhibitinteractions between PSD-95 and nNOS and other glutamate receptors(e.g., kainite receptors or AMPA receptors), such as KV1-4 and GluR6.Preferred peptides inhibit interaction between PDZ domains 1 and 2 ofpostsynaptic density-95 protein (PSD-95) (human amino acid sequenceprovided by Stathakism, Genomics 44(1):71-82 (1997)) and the C-terminalPL sequence of one or more NMDA Receptor 2 subunits including the NR2Bsubunit of the neuronal N-methyl-D-aspartate receptor (Mandich et al.,Genomics 22, 216-8 (1994)). NMDAR2B has GenBank ID 4099612, a C-terminal20 amino acids FNGSSNGHVYEKLSSIESDV (SEQ ID NO:11) and a PL motif ESDV(SEQ ID NO: 12). Preferred peptides inhibit the human forms of PSD-95and human NMDAR receptors. However, inhibition can also be shown fromspecies variants of the proteins. A list of NMDA and glutamate receptorsthat can be used appears below:

TABLE 1 NMDA Receptors With PL Sequences GI or C-terminal C-terminalinternal Name Acc# 20mer sequence 4mer sequence PL? PL ID NMDAR1 307302HPTDITGPLNLSDPSVST STVV X AA216 VV (SEQ ID NO: 13) (SEQ ID NO: 27)NMDAR1-1 292282 HPTDITGPLNLSDPSVST STVV X AA216 VV (SEQ ID NO: 13)(SEQ ID NO: 27) NMDAR1-4 472845 HPTDITGPLNLSDPSVST STVV X AA216VV (SEQ ID NO: 13) (SEQ ID NO: 27) NMDAR1- 2343286 HPTDITGPLNLSDPSVSTSTVV X AA216 3b VV (SEQ ID NO: 13) (SEQ ID NO: 27) NMDAR1- 2343288HPTDITGPLNLSDPSVST STVV X AA216 4b VV (SEQ ID NO: 13) (SEQ ID NO: 27)NMDAR1-2 11038634 RRAIEREEGQLQLCSRH HRES RES (SEQ ID NO: 14) (SEQ IDNO: 28) NMDAR1-3 11038636 RRAIEREEGQLQLCSRH HRES RES (SEQ ID NO: 14)(SEQ ID NO: 28) NMDAR2C 6006004 TQGFPGPCTWRRISSLES ESEV X AA180EV (SEQ ID NO: 15) (SEQ ID NO: 29) NMDAR3 560546 FNGSSNGHVYEKLSSIES ESDVX AA34.1 DV (SEQ ID NO: 11) (SEQ ID NO: 12) NMDAR3A 17530176AVSRKTELEEYQRTSRT TCES CES (SEQ ID NO: 16) (SEQ ID NO: 30) NMDAR2B4099612 FNGSSNGHVYEKLSSIES ESDV X DV (SEQ ID NO: 11) (SEQ ID NO: 12)NMDAR2A 558748 LNSCSNRRVYKKMPSIE ESDV X AA34.2 SDV (SEQ ID NO: 17)(SEQ ID NO: 12) NMDAR2D 4504130 GGDLGTRRGSAHFSSLE ESEV XSEV (SEQ ID NO: 18) (SEQ ID NO: 29) Glutamate AF009014 QPTPTLGLNLGNDPDRGGTSI X receptor TSI (SEQ ID NO: 19) (SEQ ID delt 2 NO: 31) GlutamateI28953 MQSIPCMSHSSGMPLGA ATGL (SEQ X receptor 1 TGL (SEQ ID NO: 20)ID NO: 32) Glutamate L20814 QNFATYKEGYNVYGIES SVKI (SEQ ID X receptor 2VKI (SEQ ID NO: 21) NO: 33) Glutamate AF167332 QNYATYREGYNVYGTESVKI (SEQ ID X receptor 3 SVKI (SEQ ID NO: 22) NO: 33) Glutamate U16129HTGTAIRQSSGLAVIASD SDLP (SEQ ID receptor 4 LP (SEQ ID NO: 23) NO: 34)Glutamate U16125 SFTSILTCHQRRTQRKET ETVA (SEQ X receptor 5VA (SEQ ID NO: 24) ID NO: 35) Glutamate U16126 EVINMHTFNDRRLPGKEETMA (SEQ X receptor 6 TMA (SEQ ID NO: 25) ID NO: 36)

Some peptides inhibit interactions between PSD-95 and multiple NMDARsubmits. In such instances, use of the peptide does not necessarilyrequire an understanding of the respective contributions of thedifferent NMDARs to excitatory neurotransmission. Other peptides arespecific for a single NMDAR.

Peptides can include or be based on a PL motif from the C-terminus ofany of the above subunits and have an amino acid sequence comprising[S/T]-X-[V/L]. This sequence preferably occurs at the C-terminus of thepeptides of the invention. Preferred peptides have an amino acidsequence comprising [E/D/N/Q]-[S/T]-[D/E/Q/N]-[V/L] (SEQ ID NO:38) attheir C-terminus. Exemplary peptides comprise: ESDV (SEQ ID NO:12), ESEV(SEQ ID NO:29), ETDV (SEQ ID NO:39), ETEV (SEQ ID NO:40), DTDV (SEQ IDNO:41), and DTEV (SEQ ID NO:42) as the C-terminal amino acids. Twoparticularly preferred peptides are KLSSIESDV (SEQ ID NO:5), andKLSSIETDV (SEQ ID NO:43). Such peptides usually have 3-25 amino acids(without an internalization peptide), peptide lengths of 5-10 aminoacids, and particularly 9 amino acids (also without an internalizationpeptide) are preferred. In some such peptides, all amino acids are fromthe C-terminus of an NMDA receptor (not including amino acids from aninternalization peptide).

Other peptides that inhibit interactions between PDS95 and NDMARsinclude peptides from PDZ domain 1 and/or 2 of PSD-95 or a subfragmentof any of these that inhibits interactions between PSD-95 and an NMDAreceptor, such as NR2B. Such active peptides comprise at least 50, 60,70, 80 or 90 amino acids from PDZ domain 1 and/or PDZ domain 2 ofPSD-95, which occur within approximately amino acids 65-248 of PSD-95provided by Stathakism, Genomics 44(1):71-82 (1997) (human sequence) orNP_031890.1, GI:6681195 (mouse sequence) or corresponding regions ofother species variants.

Peptides and peptidomimetics of the invention can contain modified aminoacid residues for example, residues that are N-alkylated. N-terminalalkyl modifications can include e.g., N-Methyl, N-Ethyl, N-Propyl,N-Butyl, N-Cyclohexylmethyl, N-Cyclyhexylethyl, N-Benzyl, N-Phenylethyl,N-phenylpropyl, N-(3,4-Dichlorophenyl)propyl,N-(3,4-Difluorophenyl)propyl, and N-(Naphthalene-2-yl)ethyl).

Bach, J. Med. Chem. 51, 6450-6459 (2008) and WO 2010/004003 havedescribed a series of analogs of NR2B9c (SEQ ID NO:6). PDZ-bindingactivity is exhibited by peptides having only three C-terminal aminoacids (SDV). Bach also reports analogs having an amino acid sequencecomprising or consisting of X₁tSX₂V (SEQ ID NO:68), wherein t and S arealternative amino acids, X₁ is selected from among E, Q, and A, or ananalogue thereof, X₂ is selected from among A, Q, D, N, N-Me-A, N-Me-Q,N-Me-D, and N-Me-N or an analog thereof. Optionally the peptide isN-alkylated in the P3 position (third amino acid from C-terminus, i.e.position occupied by tS). The peptide can be N-alkylated with acyclohexane or aromatic substituent, and further comprises a spacergroup between the substituent and the terminal amino group of thepeptide or peptide analogue, wherein the spacer is an alkyl group,preferably selected from among methylene, ethylene, propylene andbutylene. The aromatic substituent can be a naphthalen-2-yl moiety or anaromatic ring substituted with one or two halogen and/or alkyl group.

Other modifications can also be incorporated without adversely affectingthe activity and these include substitution of one or more of the aminoacids in the natural L-isomeric form with amino acids in the D-isomericform. Thus, any amino acid naturally occurring in the L-configuration(which can also be referred to as the R or S, depending upon thestructure of the chemical entity) can be replaced with the amino acid ofthe same chemical structural type or a peptidomimetic, but of theopposite chirality, generally referred to as the D-amino acid, but whichcan additionally be referred to as the R- or S-form. Thus, apeptidomimetic may include 1, 2, 3, 4, 5, at least 50%, or all D-aminoacid resides. A peptidomimetic containing some or all D residues issometimes referred to an “inverso” peptide.

Peptidomimetics also include retro peptides. A retro peptide has areverse amino acid sequence. Peptidomimetics also include retro inversopeptides in which the order of amino acids is reversed from so theoriginally C-terminal amino acid appears at the N-terminus and D-aminoacids are used in place of L-amino acids. WO 2008/014917 describes aretro-inverso analog of Tat-NR2B9c having the amino acid sequencevdseisslk-rrrqrrkkrgyin (SEQ ID NO:8) (lower case letters indicating Damino acids), and reports it to be effective inhibiting cerebralischemia. Another effect peptide described herein is Rv-Tat-NR2B9c(RRRQRRKKRGYKLSSIESDV; SEQ ID NO:70).

A linker, e.g., a polyethylene glycol linker, can be used to dimerizethe active moiety of the peptide or the peptidomimetic to enhance itsaffinity and selectivity towards proteins containing tandem PDZ domains.See e.g., Bach et al., (2009) Angew. Chem. Int. Ed. 48:9685-9689 and WO2010/004003. A PL motif-containing peptide is preferably dimerized viajoining the N-termini of two such molecules, leaving the C-termini free.Bach further reports that a pentamer peptide IESDV (SEQ ID NO:71) fromthe C-terminus of NMDAR 2B was effective in inhibiting binding of NMDAR2B to PSD-95. IETDV (SEQ ID NO:73) can also be used instead of IESDV.Optionally, about 2-10 copies of a PEG can be joined in tandem as alinker. Optionally, the linker can also be attached to aninternalization peptide or lipidated to enhance cellular uptake.Examples of illustrative dimeric inhibitors are shown below (see Bach etal, PNAS 109 (2012) 3317-3322). Any of the PSD-95 inhibitors disclosedherein can be used instead of IETDV, and any internalization peptide orlipidating moiety can be used instead of tat. Other linkers to thatshown can also be used.

IETAV is assigned SEQ ID NO:74, YGRKKRRQRRR SEQ ID NO:2, and rrrqrrkkr,SEQ ID NO:75, lower case letters indicated D-amino acids.

Appropriate pharmacological activity of peptides, peptidomimetics orother agent can be confirmed if desired, using previously described ratmodels of stroke before testing in the primate and clinical trialsdescribed in the present application. Peptides or peptidomimetics canalso be screened for capacity to inhibit interactions between PSD-95 andNMDAR 2B using assays described in e.g., US 20050059597, which isincorporated by reference. Useful peptides typically have IC50 values ofless than 50 M, 25 M, 10 JAM, 0.1 μM or 0.01 μM in such an assay.Preferred peptides typically have an IC50 value of between 0.001-1 M,and more preferably 0.001-0.05, 0.05-0.5 or 0.05 to 0.1 μM. When apeptide or other agent is characterized as inhibiting binding of oneinteraction, e.g., PSD-95 interaction to NMDAR2B, such description doesnot exclude that the peptide or agent also inhibits another interaction,for example, inhibition of PSD-95 binding to nNOS.

Peptides such as those just described can optionally be derivatized(e.g., acetylated, phosphorylated, myristoylated, geranylated and/orglycosylated) to improve the binding affinity of the inhibitor, toimprove the ability of the inhibitor to be transported across a cellmembrane or to improve stability. As a specific example, for inhibitorsin which the third residue from the C-terminus is S or T, this residuecan be phosphorylated before use of the peptide.

Pharmacological agents also include small molecules that inhibitinteractions between PSD-95 and NMDAR 2B, and/or other interactionsdescribed above. Suitable small-molecule inhibitors are described in,e.g., WO/2009/006611. An exemplary class of suitable compounds are ofthe formula:

-   -   wherein R¹ is a member selected from the group consisting of        cyclohexyl substituted with 0-4 R⁷, phenyl substituted with 0-4        R⁷, —(CH₂)_(u)—(CHR8R⁹), a branched C₁₋₆ alkyl (isopropyl,        isobutyl, 1-isopropyl-2-methyl-butyl, 1 ethyl-propyl), and        —NH—C(O)—(CR¹⁰R¹¹)_(v)H;    -   each R⁷ is independently a member selected from the group        consisting of C₁₋₆ alkyl, C₁₋₆ alkoxy, —C(O)R¹², OH, COOH, —NO,        N-substituted indoline and a cell membrane translocation        peptide;    -   each R⁸ and R⁹ is independently selected from the group        consisting of H, OH, cyclohexane, cyclopentane, phenyl,        substituted phenyl and cyclopentadiene;    -   each R¹⁰ and R¹¹ is independently selected from the group        consisting of H, cyclohexane, phenyl and a cell membrane        translocation peptide;    -   R¹² is a member selected from the group consisting of C₁₋₆ alkyl        and aryl; and each of u and v are independently from 0 to 20;    -   wherein one of R², R³, R⁴, R⁵ and R⁶ is —COOH, and wherein the        remainder of R², R³, R⁴, R⁵ and R⁶ are each independently        selected from the group consisting of F, H, OCH₃ and CH₃.

One such compound is 0620-0057, the structure of which is:

A pharmacological agent can be linked to an internalization peptide tofacilitate uptake into cells and/or across the blood brain barrier.Internalization peptides are a well-known class of relatively shortpeptides that allow many cellular or viral proteins to traversemembranes. Internalization peptides, also known as cell membranetransduction peptides or cell penetrating peptides can have e.g., 5-30amino acids. Such peptides typically have a cationic charge from anabove normal representation (relative to proteins in general) ofarginine and/or lysine residues that is believed to facilitate theirpassage across membranes. Some such peptides have at least 5, 6, 7 or 8arginine and/or lysine residues. Examples include the antennapediaprotein (Bonfanti, Cancer Res. 57, 1442-6 (1997)) (and variantsthereof), the tat protein of human immunodeficiency virus, the proteinVP22, the product of the UL49 gene of herpes simplex virus type 1,Penetratin, SynB1 and 3, Transportan, Amphipathic, gp41NLS, polyArg, andseveral plant and bacterial protein toxins, such as ricin, abrin,modeccin, diphtheria toxin, cholera toxin, anthrax toxin, heat labiletoxins, and Pseudomonas aeruginosa exotoxin A (ETA). Other examples aredescribed in the following references (Temsamani, Drug Discovery Today,9(23):1012-1019, 2004; De Coupade, Biochem J., 390:407-418, 2005; SaalikBioconjugate Chem. 15: 1246-1253, 2004; Zhao, Medicinal Research Reviews24(1):1-12, 2004; Deshayes, Cellular and Molecular Life Sciences62:1839-49, 2005); Gao, ACS Chem. Biol 2011, 6, 484-491, SG3(RLSGMNEVLSFRWL) (SEQ ID NO:77) (all incorporated by reference).

A preferred internalization peptide is tat from the HIV virus. A tatpeptide reported in previous work comprises or consists of the standardamino acid sequence YGRKKRRQRRR (SEQ ID NO:2) found in HIV Tat protein.If additional residues flanking such a tat motif are present (beside thepharmacological agent) the residues can be for example natural aminoacids flanking this segment from a tat protein, spacer or linker aminoacids of a kind typically used to join two peptide domains, e.g., gly(ser)₄ (SEQ ID NO:44), TGEKP (SEQ ID NO:45), GGRRGGGS (SEQ ID NO:46), orLRQRDGERP (SEQ ID NO:47) (see, e.g., Tang et al. (1996), J. Biol. Chem.271, 15682-15686; Hennecke et al. (1998), Protein Eng. 11, 405-410)), orcan be any other amino acids that do not significantly reduce capacityto confer uptake of the variant without the flanking residues.Preferably, the number of flanking amino acids other than an activepeptide does not exceed ten on either side of YGRKKRRQRRR (SEQ ID NO:2).One suitable tat peptide comprising additional amino acid residuesflanking the C-terminus of YGRKKRRQRRR (SEQ ID NO:2) is YGRKKRRQRRRPQ(SEQ ID NO:48). However, preferably, no flanking amino acids arepresent. Other tat peptides that can be used include GRKKRRQRRRPQ (SEQID NO:4) and GRKKRRQRRRP (SEQ ID NO:72).

Variants of the above tat peptide having reduced capacity to bind toN-type calcium channels are described by WO/2008/109010. Such variantscan comprise or consist of an amino acid sequence XGRKKRRQRRR (SEQ IDNO:49), in which X is an amino acid other than Y or nothing (in whichcase G is a free N-terminal residue). A preferred tat peptide has theN-terminal Y residue substituted with F. Thus, a tat peptide comprisingor consisting of FGRKKRRQRRR (SEQ ID NO:3) is preferred. Anotherpreferred variant tat peptide consists of GRKKRRQRRR (SEQ ID NO:1).Another preferred tat peptide comprises or consists of RRRQRRKKRG (SEQID NO:10) or RRRQRRKKRGY (SEQ ID NO: 26) (amino acidsl-10 or 1-11 of SEQID NO:70). Other tat derived peptides that facilitate uptake of apharmacological agent without inhibiting N-type calcium channels includethose presented in Table 2 below.

TABLE 2 X-FGRKKRRQRRR (F-Tat) (SEQ ID NO: 69)X-GKKKKKQKKK (SEQ ID NO: 50) X-RKKRRQRRR (SEQ ID NO: 51)X-GAKKRRQRRR (SEQ ID NO: 52) X-AKKRRQRRR (SEQ ID NO: 53)X-GRKARRQRRR (SEQ ID NO: 54) X-RKARRQRRR (SEQ ID NO: 55)X-GRKKARQRRR (SEQ ID NO: 56) X-RKKARQRRR (SEQ ID NO: 57)X-GRKKRRQARR (SEQ ID NO: 58) X-RKKRRQARR (SEQ ID NO: 59)X-GRKKRRQRAR (SEQ ID NO: 60) X-RKKRRQRAR (SEQ ID NO: 61)X-RRPRRPRRPRR (SEQ ID NO: 62) X-RRARRARRARR (SEQ ID NO: 63)X-RRRARRRARR (SEQ ID NO: 64) X-RRRPRRRPRR (SEQ ID NO: 65)X-RRPRRPRR (SEQ ID NO: 66) X-RRARRARR (SEQ ID NO: 67)

X can represent a free amino terminus, one or more amino acids, or aconjugated moiety. Internalization peptides can be used in inverso orretro or inverso retro form with or without the linked peptide orpeptidomimetic being in such form. For example, a preferred chimericpeptide has an amino acid sequence comprising or consisting ofRRRQRRKKRGY-KLSSIESDV (SEQ ID NO:70, also known as NA-1 or Tat-NR2B9c)or having an amino acid sequence comprising or consisting ofRRRQRRKKRGY-KLSSIETDV (SEQ ID NO:37).

Internalization peptides can be attached to pharmacological agents byconventional methods. For example, the agents can be joined tointernalization peptides by chemical linkage, for instance via acoupling or conjugating agent. Numerous such agents are commerciallyavailable and are reviewed by S. S. Wong, Chemistry of ProteinConjugation and Cross-Linking, CRC Press (1991). Some examples ofcross-linking reagents include J-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) or N,N′-(1,3-phenylene) bismaleinide;N,N′-ethylene-bis-(iodoacetamide) or other such reagent having 6 to 11carbon methylene bridges (which relatively specific for sulfhydrylgroups); and 1,5-difluoro-2,4-dinitrobenzene (which forms irreversiblelinkages with amino and tyrosine groups). Other cross-linking reagentsinclude p,p′-difluoro-m, m′-dinitrodiphenylsulfone (which formsirreversible cross-linkages with amino and phenolic groups); dimethyladipimidate (which is specific for amino groups);phenol-1,4-disulfonylchloride (which reacts principally with aminogroups); hexamethylenediisocyanate or diisothiocyanate, orazophenyl-p-diisocyanate (which reacts principally with amino groups);glutaraldehyde (which reacts with several different side chains) anddisdiazobenzidine (which reacts primarily with tyrosine and histidine).

For pharmacological agents that are peptides attachment to aninternalization peptide can be achieved by generating a fusion proteincomprising the peptide sequence fused, preferably at its N-terminus, toan internalization peptide.

Instead of or as well as linking a peptide (or other agent) inhibitingPSD-95 to an internalization peptide, such a peptide can be linked to alipid (lipidation) to increase hydrophobicity of the conjugate relativeto the peptide alone and thereby facilitate passage of the linkedpeptide across cell membranes and/or across the brain barrier.Lipidation is preferably performed on the N-terminal amino acid but canalso be performed on internal amino acids, provided the ability of thepeptide to inhibit interaction between PSD-95 and NMDAR 2B is notreduced by more than 50%. Preferably, lipidation is performed on anamino acid other than one of the four most C-terminal amino acids.Lipids are organic molecules more soluble in ether than water andinclude fatty acids, glycerides and sterols. Suitable forms oflipidation include myristoylation, palmitoylation or attachment of otherfatty acids preferably with a chain length of 10-20 carbons, such aslauric acid and stearic acid, as well as geranylation,geranylgeranylation, and isoprenylation. Lipidations of a type occurringin posttranslational modification of natural proteins are preferred.Lipidation with a fatty acid via formation of an amide bond to thealpha-amino group of the N-terminal amino acid of the peptide is alsopreferred. Lipidation can be by peptide synthesis including aprelipidated amino acid, be performed enzymatically in vitro or byrecombinant expression, by chemical crosslinking or chemicalderivatization of the peptide. Amino acids modified by myristoylationand other lipid modifications are commercially available.

Lipidation preferably facilitates passage of a linked peptide (e.g.,KLSSIESDV (SEQ ID NO:5), or KLSSIETDV (SEQ ID NO:43)) across a cellmembrane and/or the blood brain barrier without causing a transientreduction of blood pressure as has been found when a standard tatpeptide is administered at high dosage (e.g., at or greater than 3mg/kg), or at least with smaller reduction that than the same peptidelinked to a standard tat peptide.

Pharmacologic peptides, optionally fused to tat peptides, can besynthesized by solid phase synthesis or recombinant methods.Peptidomimetics can be synthesized using a variety of procedures andmethodologies described in the scientific and patent literature, e.g.,Organic Syntheses Collective Volumes, Gilman et al (Eds) John Wiley &Sons, Inc., NY, al-Obeidi (1998) Mol. Biotechnol 9:205-223; Hruby (1997)Curr. Opin. Chem. Biol. 1:114-119; Ostergaard (1997) Mol Divers.3:17-27; Ostresh (1996) Methods Enzymol. 267:220-234.

III. Agents and Methods for Reperfusion

Plaques and blood clots (also known as emboli) causing ischemia can bedissolved, removed or bypassed by both pharmacological and physicalmeans. The dissolving, removal of plaques and blood clots and consequentrestoration of blood flow is referred to as reperfusion. One class ofagents acts by thrombolysis. These agents work by stimulatingfibrinolysis by plasmin through infusion of tissue plasminogenactivators (tPA). Plasmin clears cross-linked fibrin mesh (the backboneof a clot), making the clot soluble and subject to further proteolysisby other enzymes, and restores blood flow in occluded blood vessels.Examples of thrombolytic agents include tissue plasminogen activatort-PA, alteplase (Activase), reteplase (Retavase), tenecteplase (TNKase),anistreplase (Eminase), streptokinase (Kabikinase, Streptase), andurokinase (Abbokinase).

Another class of drugs that can be used for reperfusion is vasodilators.These drugs act by relaxing and opening up blood vessels thus allowingblood to flow around an obstruction. Some examples of types ofvasodilators alpha-adrenoceptor antagonists (alpha-blockers),Angiotensin receptor blockers (ARBs), Beta₂-adrenoceptor agonists(β₂-agonists), calcium-channel blockers (CCBs), centrally actingsympatholytics, direct acting vasodilators, endothelin receptorantagonists, ganglionic blockers, nitrodilators, phosphodiesteraseinhibitors, potassium-channel openers, and renin inhibitors.

Another class of drugs that can be used for reperfusion is hypertensivedrugs (i.e., drugs raising blood pressure), such as epinephrine,phenylephrine, pseudoephedrine, norepinephrine; norephedrine;terbutaline; salbutamol; and methylephedrine. Increased perfusionpressure can increase flow of blood around an obstruction.

Mechanical methods of reperfusion include angioplasty, catheterization,and artery bypass graft surgery, stenting, embolectomy, orendarterectomy. Such procedures restore plaque flow by mechanicalremoval of a plaque, holding a blood vessel open, so blood can flowaround a plaque or bypassing a plaque.

Other mechanical methods of reperfusion include use of a device thatdiverts blood flow from other areas of the body to the brain. An exampleis a catheter partially occluding the aorta, such as the CoAxiaNeuroFlo™ catheter device, which has recently been subjected to arandomized trial and may get FDA approval for stroke treatment. Thisdevice has been used on subjects presenting with stroke up to 14 hoursafter onset of ischemia.

Use of a non-thromolytic agent or mechanical method of reperfusion doesnot subject a peptide PSD-95 inhibitor to proteolytic cleavage andtherefore, which may be an advantage if the PSD-95 inhibitor andreperfusion are administered simultaneously or proximate in time.

IV. Stroke

A stroke is a condition resulting from impaired blood flow in the CNSregardless of cause. Potential causes include embolism, hemorrhage andthrombosis. Some neuronal cells die immediately as a result of impairedblood flow. These cells release their component molecules includingglutamate, which in turn activates NMDA receptors, which raiseintracellular calcium levels, and intracellular enzyme levels leading tofurther neuronal cell death (the excitotoxicity cascade). The death ofCNS tissue is referred to as infarction. Infarction Volume (i.e., thevolume of dead neuronal cells resulting from stroke in the brain) can beused as an indicator of the extent of pathological damage resulting fromstroke. The symptomatic effect depends both on the volume of aninfarction and where in the brain it is located. Disability index can beused as a measure of symptomatic damage, such as the Rankin StrokeOutcome Scale (Rankin, Scott Med J; 2:200-15 (1957)) and the BarthelIndex. The Rankin Scale is based on assessing directly the globalconditions of a subject as follows.

TABLE 3 0 No symptoms at all 1 No significant disability despitesymptoms; able to carry out all usual duties and activities. 2 Slightdisability; unable to carry out all previous activities but able to lookafter own affairs without assistance. 3 Moderate disability requiringsome help, but able to walk without assistance 4 Moderate to severedisability; unable to walk without assistance and unable to attend toown bodily needs without assistance. 5 Severe disability; bedridden,incontinent, and requiring constant nursing care and attention.

The Barthel Index is based on a series of questions about the subject'sability to carry out 10 basic activities of daily living resulting in ascore between 0 and 100, a lower score indicating more disability(Mahoney et al., Maryland State Medical Journal 14:56-61 (1965)).

Alternatively stroke severity/outcomes can be measured using the NIHstroke scale, available at world wide webninds.nih.gov/doctors/NIH_Stroke_Scale_Booklet.pdf.

The scale is based on the ability of a subject to carry out 11 groups offunctions that include assessments of the subject's level ofconsciousness, motor, sensory and language functions.

An ischemic stroke refers more specifically to a type of stroke that iscaused by blockage of blood flow to the brain. The underlying conditionfor this type of blockage is most commonly the development of fattydeposits lining the vessel walls. This condition is calledatherosclerosis. These fatty deposits can cause two types ofobstruction. Cerebral thrombosis refers to a thrombus (blood clot) thatdevelops at the clogged part of the vessel “Cerebral embolism” refersgenerally to a blood clot or atheroma that forms at another location inthe circulatory system, usually the heart and large arteries of theupper chest and neck. A portion of the blood clot and/or atheroma thenbreaks loose, enters the bloodstream and travels through the brain'sblood vessels until it reaches vessels too small to let it pass. Asecond important cause of embolism is an irregular heartbeat, known asarterial fibrillation. It creates conditions in which clots can form inthe heart, dislodge and travel to the brain. Additional potential causesof ischemic stroke are hemorrhage, thrombosis, dissection of an arteryor vein, a cardiac arrest, shock of any cause including hemorrhage, andiatrogenic causes such as direct surgical injury to brain blood vesselsor vessels leading to the brain or cardiac surgery. Ischemic strokeaccounts for about 83 percent of all cases of stroke.

Transient ischemic attacks (TIAs) are minor or warning strokes. In aTIA, conditions indicative of an ischemic stroke are present and thetypical stroke warning signs develop. However, the obstruction (bloodclot) occurs for a short time and tends to resolve itself through normalmechanisms. Subjects undergoing heart surgery are at particular risk oftransient cerebral ischemic attack.

Hemorrhagic stroke accounts for about 17 percent of stroke cases. Itresults from a weakened vessel that ruptures and bleeds into thesurrounding brain. The blood accumulates and compresses the surroundingbrain tissue. The two general types of hemorrhagic strokes areintracerebral hemorrhage and subarachnoid hemorrhage. Hemorrhagic strokeresult from rupture of a weakened blood vessel ruptures. Potentialcauses of rupture from a weakened blood vessel include a hypertensivehemorrhage, in which high blood pressure causes a rupture of a bloodvessel, or another underlying cause of weakened blood vessels such as aruptured brain vascular malformation including a brain aneurysm,arteriovenous malformation (AVM) or cavernous malformation. Hemorrhagicstrokes can also arise from a hemorrhagic transformation of an ischemicstroke which weakens the blood vessels in the infarct, or a hemorrhagefrom primary or metastatic tumors in the CNS which contain abnormallyweak blood vessels. Hemorrhagic stroke can also arise from iatrogeniccauses such as direct surgical injury to a brain blood vessel. Ananeurysm is a ballooning of a weakened region of a blood vessel. If leftuntreated, the aneurysm may continue to weaken until it ruptures andbleeds into the brain. An arteriovenous malformation (AVM) is a clusterof abnormally formed blood vessels. A cavernous malformation is a venousabnormality that can cause a hemorrhage from weakened venous structures.Any one of these vessels can rupture, also causing bleeding into thebrain. Hemorrhagic stroke can also result from physical trauma.Hemorrhagic stroke in one part of the brain can lead to ischemic strokein another through shortage of blood lost in the hemorrhagic stroke.

V. Subjects Amenable to Treatment

Subjects amenable to treatment include subjects presenting with signs(s)and/or symptom(s) of ischemia either in the CNS or elsewhere in the bodybut affecting a blood vessel whose obstruction may impede blood flowthrough the brain. These subjects include subjects presenting withsign(s) and/or symptoms of stroke, myocardial ischemia, pulmonaryembolism, limb ischemia, renal or retinal ischemia. Such subjectsinclude subjects in which such a condition is suspected but otherconditions cannot be excluded, as well as subjects who have beendiagnosed according to generally recognized criteria, e.g., DSM IV TR.

Subjects amenable to treatment also include subjects at risk of ischemiabut in which onset of ischemia has not yet occurred. A subject is atrisk if he or she has a higher risk of developing ischemia than acontrol population. The control population may include one or moreindividuals selected at random from the general population (e.g.,matched by age, gender, race and/or ethnicity) who have not beendiagnosed or have a family history of the disorder. A subject can beconsidered at risk for a disorder if a “risk factor” associated withthat disorder is found to be associated with that subject. A risk factorcan include any activity, trait, event or property associated with agiven disorder, for example, through statistical or epidemiologicalstudies on a population of subjects. A subject can thus be classified asbeing at risk for a disorder even if studies identifying the underlyingrisk factors did not include the subject specifically. For example, asubject undergoing heart surgery is at risk of transient cerebralischemic attack because the frequency of transient cerebral ischemicattack is increased in a population of subjects who have undergone heartsurgery as compared to a population of subjects who have not.

Subjects at risk of ischemia affecting the brain include thoseundergoing a surgical procedure on the brain or CNS, such asendovascular surgery, clipping, stenting or microcathetherization. Suchsubjects also include those undergoing surgery elsewhere in the bodythat affects a blood vessel supplying the brain (that is connecting thebrain to the heart, for example, carotid arteries and jugular veins) oron an artery supplying blood to the retina, kidney, spinal cord orlimbs. A preferred class of subjects are those undergoing endovascularsurgery to treat a brain aneurysm. Subjects undergoing these types ofsurgery are at enhanced risk of ischemia affects the CNS. Subjects atrisk of stroke also include patients who are smokers, hypertensive,diabetic, hyper-cholesterolemic. Subjects especially at a high risk arethose who have had a prior stroke, minor stroke, or transient ischemicattack.

VI. Combined Methods of Treatment

The combined methods involved administering a PSD-95 inhibitor and aform of reperfusion therapy to a subject amenable to treatment. ThePSD-95 inhibitor and reperfusion can be administered in either order orat the same time. Usually, the PSD-95 inhibitor and reperfusion areadministered at the same, overlapping or proximate times (i.e., within a15 minutes interval) or the PSD-95 inhibitor is administered first. Fortreatment of ischemias that cannot be predicted in advance, the PSD-95inhibitor can be administered as soon as possible or practical afteronset of ischemia. For example, the PSD-95 inhibitor can be administeredwithin a period of 0.5, 1, 2, 3, 4, 5, 6, 9, 12 or 24 hours after theonset of ischemia. For ischemias that can be predicted in advance, thePSD-95 inhibitor can be administered before, concurrent with or afteronset of ischemia. For example, for an ischemia resulting from surgery,the PDS95 inhibitor is sometimes routinely administered in a periodstarting 30 minutes before beginning surgery and ending 1, 2, 3, 4, 5,6, 9, 12 or 24 hours after surgery without regard to whether ischemiahas or will develop. Because the PSD-95 inhibitor is free of seriousside effects, it can be administered when stroke or other ischemicconditions are suspected without a diagnosis according to art-recognizedcriteria having been made. For example, the PSD-95 inhibitor can beadministered at the location where the stroke has occurred (e.g., in thepatients' home) or in an ambulance transporting a subject to a hospital.The PSD-95 inhibitor can also be safely administered to a subject atrisk of stroke or other ischemic conditions before onset who may or maynot actually develop the condition.

Following, or sometimes before, administration of the PSD-95 inhibitor,a subject presenting with sign(s) and/or symptom(s) of ischemia can besubject to further diagnostic assessment to determine whether thesubject has ischemia within or otherwise affecting the CNS and determinewhether the subject has or is susceptible to hemorrhage. Mostparticularly in subjects presenting with symptoms of stroke, testingattempts to distinguish whether the stroke is the result of hemorrhageor ischemia, hemorrhage accounting for about 17% of strokes. Diagnostictests can include a scan of one or more organs, such as a CAT scan, MRIor PET imaging scan or a blood test for a biomarker that suggests that astroke has occurred. Several biomarkers associated with stroke are knownincluding B-type neurotrophic growth factor, von Willebrand factor,matrix metalloproteinase-9, and monocyte chemotactic protein-1 (seeReynolds et al., Clinical Chemistry 49: 1733-1739 (2003)). The organ(s)scanned include any suspected as being the site of ischemia (e.g.,brain, heart, limbs, spine, lungs, kidney, retina) as well as anyotherwise suspect of being the source of a hemorrhage. A scan of thebrain is the usual procedure for distinguishing between ischemic andhemorrhagic stroke. Diagnostic assessment can also include taking orreviewing a subject's medical history and performing other tests.Presence of any of the following factors alone or in combination can beused in assessing whether reperfusion therapy presents an unacceptablerisk: subject's symptoms are minor or rapidly improving, subject hadseizure at onset of stroke, subject has had another stroke or serioushead trauma within the past 3 months, subject had major surgery withinthe last 14 days, subject has known history of intracranial hemorrhage,subject has sustained systolic blood pressure >185 mmHg, subject hassustained diastolic blood pressure >110 mmHg, aggressive treatment isnecessary to lower the subject's blood pressure, subject has symptomssuggestive of subarachnoid hemorrhage, subject has had gastrointestinalor urinary tract hemorrhage within the last 21 days, subject has hadarterial puncture at noncompressible site within the last 7 days,subject has received heparin with the last 48 hours and has elevatedPTT, subject's prothrombin time (PT) is >15 seconds, subject's plateletcount is <100,000 μL. subject's serum glucose is <50 mg/dL or >400mg/dL, subject is a hemophiliac or has other clotting deficiencies.

The further diagnostic investigation determines according to recognizedcriteria or at least with greater probability that before theinvestigation whether the subject has an ischemic condition, and whetherthe subject has a hemorrhage, has an unacceptable risk of hemorrhage oris otherwise excluded from receiving reperfusion therapy due tounacceptable risk of side effects. Subjects in which a diagnosis of anischemic condition within or otherwise likely to affect the CNS isconfirmed who are without unacceptable risk of side effects can then besubject to reperfusion therapy. Preferably, reperfusion therapy isperformed as soon as practical after completion of any diagnosticprocedures. In some subjects, reperfusion therapy is commenced more than0, 1, 2, 3, 4, 4.5, 5, 6, 7, 8, 10, 12, 15, 18, or 24 hr after onset ofischemia. In strokes occurring in a medical setting (e.g., duringendovascular procedures) treatment can begin less than 1 hour afteronset. In some subjects, reperfusion therapy is commenced 0.5-6, 0.5-12,0.5-18 or 0.5-24 hr after onset of ischemia. In some subjects,reperfusion therapy is commenced outside the usual 3-4.5 hr window inwhich reperfusion therapy has hitherto been considered to effective. Forexample in some subjects, reperfusion therapy is commenced more than 3hours or more than 4.5 hours after onset of ischemia and up to 24 or 48hours after onset of ischemia. In some subjects, reperfusion therapy iscommenced, after 5, 6, 7, 8, 9 or 10 hours and up to 24 or 48 hoursafter onset of ischemia. In some subjects, reperfusion therapy iscommenced from 275-390 minutes after onset of ischemia. In somesubjects, reperfusion therapy is commenced irrespective of the time ofonset of ischemia provided that they qualify for reperfusion based onspecific diagnostic criteria such as the absence of a completed infarcton a CT scan, evidence of an ischemic penumbra by computerizedtomography (CT), magnetic resonance imaging (MRI) or positron emissiontomography (PET) imaging criteria, and the absence of a brainhemorrhage.

The time of reperfusion can also be measured from the administration ofthe PSD-95 inhibitor. The interval can be, for example, 5 minutes to 24or 48 hours (the interval between PSD-95 administration and reperfusion,here as elsewhere in this application, being measured from initiatingPSD-95 inhibitor administration to initiating reperfusionadministration). The interval may be for example, 15 min to 6 hr, 15 minto 4.5 hr, 15 min to 3 hr, 15 min to 1 hr, 30 minutes to 6 hours, or 30min to 3 hours, or 30 min to 4.5 hours, or 1-3 hours, or 1-4.5 hours. Alonger interval can be advantageous for peptide PSD-95 inhibitors, suchas Tat-NR2B9c, used in combination with agents for reperfusion actingvia proteolysis (e.g., tPA), because it gives the inhibitor a longerperiod to exert its effect before it is subject to promolyticdegradation by plasmin resulting.

Subjects in which an ischemic condition is not confirmed or isconsidered unlikely are not usually administered reperfusion therapy.Such subjects may not have received any benefit from the PSD-95inhibitor but are also not worse off than not having been treated.Subjects in which an ischemic condition is confirmed or consideredlikely but are considered at unacceptable risk of side effects fromreperfusion therapy are not administered reperfusion therapy. Suchsubjects may have obtained benefit of the PSD-95 inhibitor but arespared the risk of unacceptable side effects from reperfusion therapy.

Both treatment with a PSD-95 inhibitor and reperfusion therapyindependently have ability to reduce infarction size and functionaldeficits due to ischemia. When used in combination according to thepresent methods, the reduction in infarction size and/or functionaldeficits is preferably greater than that from use of either agent aloneadministered under a comparable regime other than for the combination(i.e., co-operative). More preferably, the reduction in infarction sideand/or functional deficits is at least additive or preferably more thanadditive (i.e., synergistic) of reductions achieved by the agents aloneunder a comparable regime except for the combination. In some regimes,the reperfusion therapy is effective in reducing infarction size and/orfunctional times at a time post onset of ischemia (e.g., more than 4.5hr) when it would be ineffective but for the concurrent or prioradministration of the PSD-95 inhibitor. Put another way, when a subjectis administered a PSD-95 inhibitor and reperfusion therapy, thereperfusion therapy is preferably at least as effective as it would beif administered at an earlier time without the PSD-95 inhibitor. Thus,the PSD-95 inhibitor effectively increases the efficacy of thereperfusion therapy by reducing one or more damaging effects of ischemiabefore or as reperfusion therapy takes effects. The PSD-95 inhibitor canthus compensate for delay in administering the reperfusion therapywhether the delay be from delay in the subject recognizing the danger ofhis or her initial symptoms delays in transporting a subject to ahospital or other medical institution or delays in performing diagnosticprocedures to establish presence of ischemia and/or absence ofhemorrhage or unacceptable risk thereof. Statistically significantcombined effects of PSD-95 inhibitor and reperfusion therapy includingadditive or synergistic effects can be demonstrated between populationsin a clinical trial or between populations of animal models inpreclinical work.

VI. Effective Regimes of Administration

A PSD-95 inhibitor is administered in an amount, frequency and route ofadministration effective to reduce, inhibit or delay one or moredamaging effects of ischemia on the CNS. Unless otherwise indicated,dosages for inhibitors that are chimeric agents including apharmacologic agent linked to an internalization peptide refer to thewhole agent rather than just the pharmacological agent component of thechimeric agent. An effective amount means an amount of agent sufficientsignificantly to reduce, inhibit or delay one or more damaging effectsof ischemia in a population of subjects (or animal models) sufferingfrom the disease treated with an agent of the invention relative to thedamage in a control population of subjects (or animal models) sufferingfrom that disease or condition who are not treated with the agent. Theamount is also considered effective if an individual treated subjectachieves an outcome more favorable than the mean outcome in a controlpopulation of comparable subjects not treated by methods of theinvention. An effective regime involves the administration of aneffective dose at a frequency and route of administration needed toachieve the intended purpose.

When the condition requiring treatment is stroke, the outcome can bedetermined by infarction volume or disability index, and a dosage can berecognized as effective if an individual treated subject shows adisability of two or less on the Rankin scale and 75 or more on theBarthel scale, see Lees et at L, N Engl J Med 2006; 354:588-600 or if apopulation of treated subjects shows a significantly improved (i.e.,less disability) distribution of scores on any disability scale (e.g.,Barthel, Rankin, NIH Stroke Scale) than a comparable untreatedpopulation, or if a population of treated subjects shows significantlyreduced infarction size or number compared with a comparable untreatedpopulation. A single dose of agent is usually sufficient for treatmentof stroke. However, multiple dosages can be administered at intervals ofe.g., 1, 2, 3, 6, 12, 18, or 24 hours until presence of a completedinfarct is detected on a CT scan or until no further benefit is seen.

Depending on the agent, administration can be parenteral, intravenous,nasal, oral, subcutaneous, intra-arterial, intracranial, intrathecal,intraperitoneal, topical, intranasal or intramuscular. Intravenousadministration is preferred for peptide agents.

For chimeric agents including an internalization peptide, particularly aHIV tat peptide comprising the amino acid sequence YGRKKRRQRRR (SEQ IDNO:2), administration of the agent may or may not be combined with ananti-inflammatory agent to reduce release of histamine and itsdownstream effects associated with high levels of the internalizationpeptide. Preferred agents for co-administration are inhibitors of mastcell degranulation, such as cromolyn or lodoxamide or any others listedherein. Anti-histamines or corticosteroids can also be used,particularly in combinations or higher dosages (see WO2009/076105 andWO2010/144742).

For administration to humans, a preferred dose of the chimeric agentTat-NR2B9c is 2-3 mg/kg and more preferably 2.6 mg/kg. Indicated dosagesshould be understood as including the margin of error inherent in theaccuracy with which dosages can be measured in a typical hospitalsetting. The dose is preferred because it is the maximum dose with whichthe agent can be administered without release of significant amounts ofhistamine and the ensuing sequelae in most subjects. Although release ofhistamine at higher dosages can be controlled by co-administration of ananti-inflammatory as discussed above and in any event usuallyspontaneously resolves without adverse events, it can best be avoided bykeeping the dose below 3 mg/kg and preferably at 2-3 mg/kg, morepreferably 2.6 mg/kg. Such amounts are for single dose administration,i.e., one dose per episode of disease. Such doses can also beadministered daily, or more frequently. Lower doses may be used,optionally 1-2 mg/kg, or 0.5-1 mg/kg, 0.1-0.5 mg/kg or less than 0.1mg/kg. For repeated dose regimes, even lower dosages may be used.

The dosages indicated above are for the chimeric agent Tat-NR2B9c(YGRKKRRQRRRKLSSIESDV; SEQ ID NO:6). Equivalent dosages for other agentsto achieve the same effect can be determined by several approaches. Forclose variants of that agent in which one or a few amino acids aresubstituted, inserted or deleted and the molecular weight remains thesame within about +/−25%, the above dosages are still a good guide.However, in general, for other agents, equivalent dosages can varydepending on the molecular weight of the agent with and withoutinternalization peptide if present, its Kd for its target, and itspharmacokinetic and pharmacodynamic parameters. For some agents,equivalent dosages can be calculated so as to deliver an equimolaramount of the pharmacological agent. For other agent, further adjustmentcan be made to account for differences in Kd or pharmacokinetic orpharmacodynamic parameters. For some agents, equivalent dosages aredetermined empirically from the dose achieved to reach the same endpointin an animal model or a clinical trial.

Peptide agents, such as Tat-NR2B9c are preferably delivered by infusioninto a blood vessel, more preferably by intravenous infusion. For thechimeric agent Tat-NR2B9c, a preferred infusion time providing a balancebetween these considerations is 5-15 minutes and more preferably 10minutes. Indicated times should be understood as including a marking oferror of +/−10%. Infusion times do not include any extra time for a washout diffusion to wash out any remaining droplets from an initialdiffusion that has otherwise proceeded to completion. The infusion timesfor Tat-NR2B9c can also serve as a guide for other pharmacologicalagents, optionally linked to internalization peptides, particularlyclose variants of Tat-NR2B9c, as discussed above.

The PSD-95 inhibitor can be administered in the form of a pharmaceuticalcomposition. Pharmaceutical compositions are typically manufacturedunder GMP conditions. Pharmaceutical compositions for parenteraladministration are preferentially sterile (e.g., filter sterilization ofpeptide) and free of pyrogens. Pharmaceutical compositions can beprovided in unit dosage form (i.e., the dosage for a singleadministration). Pharmaceutical compositions can be formulated inconventional manner using one or more physiologically acceptablecarriers, diluents, excipients or auxiliaries that facilitate processingof chimeric agents into preparations which can be used pharmaceutically.Proper formulation is dependent on the route of administration chosen.

An exemplary formulation of the chimeric agent Tat-NR2B9c contains thepeptide in normal saline (0.8-1.0% and preferably 0.9% saline) orphosphate buffered saline at a concentration of 10-30 mg/ml, for example16-20 or 18 mg/ml or 20 mg/ml. When stored frozen, such a composition isstable (insignificant degradation or aggregation of the peptide) for aperiod of four or more years. Although additional excipients can beadded, normal saline or phosphate buffered saline without suchexcipients is sufficient to obtain this stability. For use such acomposition is thawed and diluted into a larger volume of normal salinefor infusion into a blood vessel.

Many examples of pharmacological agent for reperfusion are in clinicaluse. Such agents can be used in the present combination methods inaccordance with their conventional formulations, doses, routes ofadministration, and frequency of administration (see Physician's DeskReference and applicable package inserts). For example, tPA or otherthrombolytic agents can be administered intravenously at a dose of e.g.,0.5-1.5 mg/kg, preferably 0.9 mg/kg in humans. tPA and otherthrombolytic agents can also be given intra-arterially preferably at adose of 0.02-0.1 mg/kg/hour in human patients for up to 36 hours. tPAand other thrombolytic agents can also be administered directly to asite of impaired blood flow, e.g., an emboli in the brain, for which thepreferred dose is 2 mg in human patients (or less in patient with weightless than 30 kg). Localized administration is preferably via a catheter.Direct administration to the site of infarction reduces potentialexposure of peptide PSD-95 inhibitors to proteolytic degradation.Likewise, mechanical methods of reperfusion can be employed inaccordance with conventional practice.

EXAMPLES Example 1 Neuroprotection After Stroke in Gyrencephalic OldWorld Primates

Introduction

We used higher order gyrencephalic nonhuman primates (NHPs) which beargenetic, anatomic and behavioral similarities to humans and testedneuroprotection by PSD95 inhibitors, compounds that uncouplepostsynaptic density protein PSD-95 from neurotoxic signaling pathways.Here we show that stroke damage can be prevented in NHPs in which aPSD95 inhibitor is administered after stroke onset. This treatmentreduced infarct volumes as gauged by magnetic resonance imaging (MRI)and histology, preserved the capacity of ischemic cells to maintain genetranscription in genome-wide screens of ischemic brain tissue, andsignificantly preserved neurological function in neurobehavioral assays.The degree of tissue neuroprotection by MRI corresponded strongly to thepreservation of neurological function, supporting the unproven dictumthat brain tissue integrity can reflect functional outcome. Our findingsestablish that tissue neuroprotection and improved functional outcomeafter stroke is unequivocally achievable in gyrencephalic NHPs usingPSD95 inhibitors.

General Experimental Flow and Assessments

We conducted all experiments with allocation concealment and blindedassessment of all outcomes. The primary outcome measure was infarctvolume at 30 days measured from a T2-weighted MRI study. Anatomicalsecondary outcomes were infarct volumes at 4 h and 24 h bydiffusion-weighted imaging (DWI) MRI, at 24 h by T2 MRI and at 30 d byT2 MRI and histology. Neurobehavioral outcomes were measured throughoutthe 30 d observation period using the non-human primate stroke scale(NHPSS) and a sensorimotor battery of tasks comprising the hill andvalley task, two-tube task and six well task. A scheme for thetreatments and assessments is presented in FIG. 1A, and a descriptionfollows below. The times of middle cerebral artery occlusion variedbetween experiments as described below.

Macaques were randomized to receive a 10 min intravenous infusion ofTat-NR2B9c (2.6 mg/kg) or placebo (0.9% saline) beginning either 1 hr or3 hr after the onset of a 90 min MCAO. The dose selected for NHPs wasapproximated from calculations of a “primate equivalent dose”extrapolated from prior doses used in rat studies and was based onnormalization to interspecies differences in body surface area.

Animals were transferred to the MRI scanner within 15 min of MCAO andall underwent perfusion imaging to quantify the brain volume deprived ofblood flow during MCAO (tissue-at-risk). Additionally, MR angiography(MRA) was conducted to confirm MCAO. A second MRA was performed afterthe 90 min MCAO was terminated to confirm reperfusion of the MCA,followed by diffusion imaging at 4 h. Animals were then awakened andallowed to recover. They were re-anaesthetized and re-imaged at 24 h andat 30 days. NHPSS scores were assigned within 8 h of MCAO and up to 30days, and the remaining neurological tests were conducted on days 7 andeither 14 or 30.

There were no differences in physiological parameters between groups inthe experiments, or in the volume of tissue at risk between the drug andplacebo groups as determined by perfusion imaging within 15 min of MCAO.

We conducted neurological assessments throughout the 14 to 30 dobservation periods using the non-human primate stroke scale (NHPSS)and, in some cases, a sensorimotor battery of tasks including the Hilland Valley Task, two-tube choice task and six well task. The NHPSS is acomposite of ratings analogous to the NIH Stroke scale used in humanstroke trials. A score of 41 points represents severe bilateralneurological impairment and 0 is normal. The remaining tests measure acombination of overall strength of the extremity, fine motor functionand the influence ofa hemi-neglect or visual field defect.

Tat-NR2B9c Treatment Reduces Brain Ischemia and can Extend the TimeWindow for Reperfusion Therapy Beyond the Current Useful 4.5 Hour Windowfor the Use of tPA.

Currently, the only widely approved treatment for acute ischenic strokeis reperfusion of occluded brain arteries using the intravenous infusionof the fibrinolytic agent, rt-PA (recombinant tissue plasminogenactivator). Conventional reperfusion with intravenous rt-PA is mostbeneficial in improving clinical outcomes when administered within 90min after stroke onset, and benefit decreases thereafter until it ismarginal or nil at 4.5 h. This narrow window for the utility ofreperfusion limits the number of patients who might benefit. Thus onepotential application of early treatment with a neuroprotectant is toextend the interval during which clinical benefit may be obtainable fromreperfusion therapy. Neurosurgeons and neurointerventionalists can usereperfusion therapy outside of the standard window if the condition ofthe brain indicates there is still useful brain to save and it would besafe to do so without risking a hemorrhage. To examine this, weevaluated in the NHPs whether administration of Tat-NR2B9c 60 min afterMCAO onset might improve stroke outcome when reperfusion is delayeduntil the 4.5 h time point, at which i.v. rt-PA is no longer ofsignificant benefit in humans (see also Cook et al., Nature. 483, 213-72012).

Six macaques were subjected to a permanent MCAO, and were treated eitherwith Tat-NR2B9c or with placebo beginning at 5 minutes after ischemiaonset. The animals were placed in the MRI scanner, and DWI MRI scanswere obtained every 15 min (FIG. 2A). The volume of brain in which DWIhyperintensity was detectable increased over time in both groups.However, treatment with Tat-NR2B9c attenuated the rate of this increaseby about twofold (FIG. 2A; time constants=2.20±0.28 h and 4.50±0.54 forcontrol and Tat-NR2B9c, respectively; p=0.019). Even after a 4.5 hourMCAO, Tat-NR2B9c treated animals showed significant reduction in infarctsize by MRI and T2 scans at 48 hours and 7 days (FIG. 1B-D). Moreover,within the ischemic volume, the DWI intensity in brains of Tat-NR2B9ctreated animals remained lower than that of untreated controls,suggesting that tissue within the infarct volume maintained betterintegrity (FIG. 2B). These data provide evidence that there remainsbrain that can be salvaged and administration of reperfusion drugs ortherapy would be likely to improve reperfusion and survival of braintissues at times when they would not normally be considered due to beingoutside of the effective time window. They also provide evidence thatTat-NR2B9c can extend the useful time for reperfusion of the brain tosave the remaining tissue.

Tat-NR2B9c Significantly Reduces Infarct Volumes and NeurologicalDeficits in Non Human Primates Subjected to a 90 Minute Stroke.

A subsequent study looking at the effect of Tat-NR2B9c when given aftera 1 hour after an ischemic stroke was tested in this model. Twentymacaques were randomized to receive a 10 min intravenous infusion ofTat-NR2B9c (2.6 mg/kg) or placebo (0.9% saline) beginning 1 h after theonset of a 90 min MCAO. The dose selected for NHPs was approximated fromcalculations of a “primate equivalent dose” extrapolated from priordoses used in rat studies and was based on normalization to interspeciesdifferences in body surface area.

Animals were transferred to the MRI scanner within 15 min of MCAO andall underwent perfusion imaging to quantify the brain volume deprived ofblood flow during MCAO (tissue-at-risk). Additionally, MR angiography(MRA) was conducted to confirm MCAO. A second MRA was performed toconfirm reperfusion after the 90 min MCAO, followed by diffusion imagingat 4 h. Animals were then awakened and allowed to recover. They werere-anaesthetized and re-imaged at 24 h and at 30 days. NHPSS scores wereassigned within 8 h of MCAO and up to 30 days, and the remainingneurological tests were conducted on days 7 and 30.

Four of 10 animals receiving placebo died within 48 h of their strokesdue to brain swelling and uncal herniation. Three animals treated withTat-NR2B9c died as a result of surgical/anesthetic complicationsunrelated to stroke or to drug. None was excluded from the“intent-to-treat” (IT) analysis. All missing data due to earlymortalities were imputed to reflect the largest possible infarct volumesand worst neurological scores. Although this approach biases againstdetecting a significant treatment effect, it is the most conservative,and most reflective of that employed in human clinical trials.

There were no differences between the drug and placebo groups inphysiological parameters or in the volume of tissue at risk asdetermined by perfusion imaging within 15 min of MCAO (FIG. 6A).However, by 24 h, animals treated with Tat-NR2B9c exhibited asignificant reduction in infarct volume as compared with placebo by DWIimaging (44.0% reduction; p=0.039; FIG. 6B) and by T2-weighted imaging(37.4% reduction, p=0.010; FIG. 6B, C). This reduction in infarct volumepersisted as reflected by the 30 d T2-weighed MRI scans (38.7%reduction, p=0.013; FIG. 6C) and by histological evaluation at 30 d(FIG. 6D; 59.3% and 73.6% reduction in infarct volume when evaluated byITT and with early mortalities removed, respectively; p<0.001). BecauseNHPs, like humans, may have variable infarcts after MCAO, the infarctvolume of each animal was normalized to its MRI perfusion defectmeasured within 15 min of MCAO. This normalization revealed thattreatment with Tat-NR2B9c reduced infarcts by 55% of the volume at riskby 24 h as gauged by DWI imaging, and by 70% at 30 d as measured withT2-weighed MRI (FIG. 6E). Infarct volumes calculated from the 24 h DWIMRI correlated well with those obtained from the 30 d histologicalanalysis (R=0.691, p<0.01).

We conducted neurological assessments throughout the 30 d observationperiod using the non-human primate stroke scale (NHPSS) and asensorimotor battery of tasks including the Hill and Valley Task,two-tube choice task and six well task. The NHPSS is a composite ofratings analogous to the NIH Stroke scale used in human stroke trials. Ascore of 41 points represents severe bilateral neurological impairmentand 0 is normal. The remaining tests measure a combination of overallstrength of the extremity, fine motor function and the influence of ahemi-neglect or visual field defect.

Animals treated with Tat-NR2B9c exhibited improved NHPSS scores from theearliest assessment at 8 h post-ischemia onset and throughout the 30 dobservation period (P=0.018, Two way repeated measures ANOVA; FIG. 6F).Performance in the 2 tube choice task returned to pre-stroke levels inanimals treated with Tat-NR2B9c, but remained completely impaired in theplacebo group, suggesting that brain salvage prevented “extinction”, thetendency for attention to items in ipsilesional hemispace to overshadowattention to items in contralesional hemispace. Treatment withTat-NR2B9c also significantly improved the performance of animals in the6-well and the Hill and Valley Staircase tasks in the left upperextremity. Right upper extremity performance also showed improvements,suggesting overall improved attention and perceptual ability.

Tat-NR2B9c is Effective in Reducing Infarct Size and NeurologicalDeficits in Severe Strokes

We next demonstrated the efficacy of Tat-NR2B9c in strokes that lastedlonger than the limit of effectiveness of reperfusion with intravenoustPA, 4.5 hours. Twelve macaques were randomized to receive a 10 minintravenous infusion of Tat-NR2B9c (2.6 mg/kg) or placebo (0.9% saline)beginning 1 h after the onset of a 4.5 h min MCAO. Otherwise, methodswere similar to our first study except for the timing of MRI scans andthat final imaging and neurological assessments were conducted at 7days.

There were no mortalities and no differences between the drug andplacebo groups in physiological parameters or in the volume of tissue atrisk upon MCAO (FIG. 7A). However, despite the prolonged ischemicinterval, animals treated with Tat-NR2B9c exhibited a significantreduction in infarct volumes as compared with placebo when evaluated byT2 and DWI imaging at 48 h and by T2-weighted imaging at 7 days (FIGS.7B-E). Moreover, animals treated with Tat-NR2B9c exhibited improvedNHPSS scores from the earliest assessment at 12 h post-ischemia onsetand throughout the 7D observation period (P=<0.001; Two way repeatedmeasures ANOVA; FIG. 7F), and trended to better performance in the6-well and the Valley Staircase tasks. These results suggest that earlytreatment with Tat-NR2B9c may increase the window during whichreperfusion may have functional benefits, even in the model of severeMCAO in which collateral circulation is limited and the penumbra issmall. The size of the benefit of treatment at 4.5 h post-stroke asgauged by MRI and by neurological evaluations suggest a potential forutility of early neuroprotection to extend the benefits of reperfusiontherapy even beyond the 4.5 h window.

Tat-NR2B9c is Effective at Reducing Ischemia and Neurological DeficitsFollowing Stroke when Given 3 Hours after the Onset of Stroke

Although treatment with a neuroprotectant within 60 in of stroke onsetis feasible in a small subset of patients, extending the therapeuticwindow of administration would benefit a much greater proportion ofstroke victims. Thus we determined whether administering Tat-NR2B9c at 3h after stroke onset is beneficial in the setting of a prolonged MCAO.In humans, reperfusion with i.v. rtPA administered 3 h after strokeonset is beneficial even in the absence of neuroprotection. This atteststo the existence of a salvageable penumbra at this time in manypatients. We reproduced this clinical scenario experimentally by usingthe MCAO model in which the NHPs exhibit a significant PWI/DWI mismatch(penumbra) at 3 h. Like in humans, such a mismatch progresses toinfarction in the absence of treatment.

Twenty-four macaques were randomized to receive a 10 min intravenousinfusion of Tat-NR2B9c (2.6 mg/kg) or placebo (0.9% saline) beginning 3h after the onset of a 3.5 h MCAO. Other methods were unchanged, exceptthat final imaging and neurological assessments were conducted at 14days. There were no mortalities and no differences in physiologicalparameters or in the volume of tissue at risk upon MCAO between thegroups (FIG. 8A). However, despite both the prolonged ischemic intervaland the delayed treatment with Tat-NR2B9c, drug-treated animalsexhibited significant reductions in infarct volumes as compared withplacebo as evaluated by anatomical (MRI) criteria at 48 h and 14 days(FIG. 8B-E). The NHPs treated with Tat-NR2B9c also exhibited improvedNHPSS scores throughout the 14 d observation period (P=0.004 Two wayrepealed measures ANOVA; FIG. 8F), and tended to better performance inthe 6-well and the Valley Staircase tasks. Thus, treatment withTat-NR2B9c 3 h after stroke onset is effective in reducing stroke damagein NHPs. As this therapeutic window is practical in stroke victims,treatment with a PSD95 inhibitor can constitute a clinically-practicabletherapeutic strategy, and be more effective that the use of reperfusionalone.

Conclusions

Tat-NR2B9c can effectively reduce the severity of the damage followingstroke in higher order brains like monkeys and humans. Examining the MRIDWI and T2 images at time points following the stroke demonstrates thatthe areas of ischemia are significantly less damaged than those inuntreated animals. In addition to the reduction in volumes of thelesions, this is suggested by less intense images by DWI-MRI and reducedsignal intensity on the T2 images. These data provide evidence thatthere is still brain left to save and that reperfusion therapy can stillallow blood to penetrate into and act upon a greater portion of thebrain that it would otherwise be able to without Tat-NR2B9c treatment.Thus, reperfusion therapy, especially with drugs like tPA, is able tobetter penetrate and be effective at further increasing the blood flowto the affected areas of the brain.

Non-Human Primate Methods

Stroke Model:

Animals were anesthetized (Isoflurane 1.0-2.5%), intubated andventilated. Non-invasive monitoring included BP by leg cuff, end-tidalCO2, O2 saturation, ECG and temperature by rectal probe. Temperature wasmaintained (37±0.5 C) by heating blanket. A femoral arterial line wasused to monitor BP and blood gases. MCAO in cynomolgus macaques (3.0-4.0kg) was performed using a right pterional craniotomy and occluding theright MCA in the Sylvian fissure with a 5 mm titanium aneurysm clipdistal to the orbitofrontal branch and origin of lenticulo-striatearteries.

Defining Ischemic Penumbra Tissue:

Penumbra tissue was operationally defined as tissue which is not yetinfarcted at the time of tissue harvest, but which consistently goes onto later infarction. Because the penumbra might be variable in macaques,eight 2×2 mm biopsies were taken at either 1 h or 6 h post MCAO fromcortex across the entire MCA vascular distribution ipsilateral to thestroke and also contralateral to the stroke from sites mirroring thosetaken from ischemic cortex. Biopsied positions were photographed. Todetermine which of the 8 biopsies represent penumbral tissue, theanimals were transferred to a 7T MRI scanner and diffusion weighted(DWI), T2 and perfusion imaging was performed within 15 minutes afterthe biopsy and at 5.75 h. Penumbra tissue at 1 h was defined as tissuedevoid of infarction that progressed to infarction at 5.75 h by DWI.Penumbral tissue at 6 h was defined as tissue within the confines of theMR perfusion defect but without demonstrated DWI or T2 hyperintensity.The use of MRI to define the penumbra of NHPs is essential as the amountof salvageable tissue shrinks by 6 h.

General Mri Procedures:

Imaging was performed on a 7-Tesla Bruker BioSpec system runningParavision 4.0 software and using a B-GA20S gradient coil. A 15.5 cminner diameter quadrature transmit/receive volume coil was used for NHPscans. NHPs were intubated, ventilated and imaged prone. Physiologicalmonitoring was maintained throughout. The protocol provides stacks of 2DT2-weighted, perfusion and diffusion-weighted images in an axial plane.T2-weighted imaging uses the RARE method, also termed fast-spin echo(TE/IR= 84/5000 ms, rare factor=14, 225×225 matrix over a 9 cmfield-of-view for 400×400×1500 micron resolution). Diffusion-weightedimaging uses a spin-echo multi-shot echo-planar imaging technique(TE/IR= 32/10000 ms, 9 EPI shots, 250 kHz bandwidth, 3 orthogonaldiffusion directions at b=1000 s/mm2, 10 averages with a 180×180 matrixover a 9 cm field-of view for 500×500×1500 micron resolution). Perfusionimaging was performed using a dynamic, contrast enhanced,susceptibility-weighted perfusion method (T2*EPI, TE=18 ms, 2 EPI shots,2-sec temporal resolution, and 90 repetitions, 180×180 matrix over a 9cm field of view for 700×700×1500-micron resolution over 5 contiguousslices). For perfusion scans, gadolinium (0.1 mmol/kg) bolus wasinjected intravenously, starting on the third repetition with a totalinjection time of 7-sec through a peripheral intravenous. Diffusionimages are post-processed in MATLAB (Natick, Mass., USA) to generate anaverage image from three b=1000 s/mm2 images and to calculate anApparent Diffusion Coefficient (ADC) map. Stroke volumes were calculatedusing ITK-Snap contouring software (Pittsburgh, Pa., USA) with stacks ofaverage diffusion images reconstructed in 3-dimensions. Perfusionimaging was processed using PerfTool software to produce cerebral bloodflow maps.

Experimental Design and Statistical Analysis:

The stroke experiments were performed in compliance with the“recommendations for ensuring good scientific enquiry” of the StrokeTherapy Academic Industry Roundtable (STAIR) committee. A sample size of10 animals/groups was based on the desire to detect a 40% difference ininfarct volumes between drug and placebo based on the 30 d T2 weightedMRI at a power of 0.8, alpha=0.05 and an assumed standard deviation of30% of group means. Primary analysis was based on an intent-to-treatapproach, with no exclusions of any animals enrolled. 20 cynomologusmacaques were block-randomized to treatment with drug or placebo(vehicle only). The investigators responsible for the induction,maintenance, and reversal of ischemia, for decisions regarding the careof (including the early sacrifice of) experimental animals, andassessment of all outcomes were blinded to the experimental group towhich an animal belonged. Differences between groups were measured usingStudent's t-test, or repeated-measures ANOVA, as required. Missingvalues due to premature death or inability to complete a task wereimputed to reflect the worst score achievable on the task, or themaximum possible stroke volume as defined by largest infarct volumeachieved across all animals.

Neurological Assessments:

These were conducted using the previously validated non-human primatestroke scale (NHPSS) and a sensorimotor battery of tasks including theHill and Valley Task, two-tube task and six well task. The NHPSS scoreis a composite of ratings of state of consciousness, defense reaction,grasp reflex, extremity movement, gait, circling, bradykinesia, balance,neglect, visual field cut/hemianopsia and facial weakness, many of whichare also incorporated in the NIH Stroke Scoring system in humans. From atotal of 41 points, 0 corresponds to normal behavior and 41 to severebilateral neurological impairment. The remaining tests were modifiedfrom assays developed for the common marmoset (Callithrix jacchus) asdescribed elsewhere. In additional to evaluating finer sensorimotorfunctions they also test extinction and perceptual spatialimpairment/neglect. In pilot experiments in 5 macaques subjected to a 90min MCAO, NHPSS results demonstrated an initial peak in score (mean36.3, SEM=5.7) that persisted for the first 36 hours and then graduallydropped to a plateau between 14 and 30 days (mean=14.36, SEM=3.2).Sensorimotor testing revealed that animals had severe leftspatiotemporal neglect and left hemiparesis that showed minor recoveryover time at 7 and 30 days following stroke. These deficits were evidentas significant delays in completion of 6 well (mean delay of 7.8× and5.33× baseline) and Hill and Valley tasks for the left arm (mean delayof 8.2× and 6.4× baseline on Valley segment and mean delay of 7.6× and5.8× baseline on Hill segment).

Example 2: PSD-95 Inhibitors Freeze Ischemic Penumbra Evolution onPerfusion/Diffusion Weighted MRI

The purpose of this example was to demonstrate that neuroprotectionusing a PSD-95 inhibitor is feasible without cerebral blood flowaugmentation (reperfusion) in experimental permanent MCAO.

Methods: Rats were subjected to pMCAO and were treated 1 h thereafterwith a 5 minute intravenous infusion of the PSD-95 inhibitor Tat-NR2B9c(7.5 mg/kg) or saline. Perfusion MRI (PWI) and diffusion MRI (DWI) wereobtained with a 4.7T Bruker system at 30, 45, 70, 70, 120, 150 and 180minutes post pMCAO to determine cerebral blood flow (CBF) and apparentdiffusion coefficient (ADC) maps. At 24 hours animals wereneurologically scored, sacrificed, and brains sectioned and stained withTTC to ascertain infarct volumes correct for edema. The effect ofTat-NR2B9c on ATP levels were measured in vitro in neurons subjected toOGD as described (Aarts et al. (2002), supra) and AT^(P) levels wereassessed using a CellTiter-Glo Luminescent Cell Viability Assayaccording to manufacturer's instructions (Promega, Madison, Wis.).

Results: Blood gases, electrolytes, and blood glucose did not differbetween the two groups. Neuroscores at 24 hours showed the Tat-NR2B9cgroup had a significantly improved neuroscore compared to placebo. FIG.3 indicates the absolute mismatch between CBF and ADC-derived lesionvolumes. Relative to placebo animals, the ADC/CBF mismatch lesionvolumes were significantly larger starting at 90 minutes after occlusionin the Tat-NR2B9C group. The region of interest analysis of the relativeCBF values in the core and cortical penumbra regions showed nosignificant change in relative CBF between time points in eithertreatment group, indicating no effect of Tat-NR2B9c treatment on CBF.

The spatiotemporal evolution of the ischemic stroke described bythreshold-derived ADC and CBF lesion volumes, correlated well to theTTC-derived lesion volumes in placebo treated animals. In Tat-NR2B9ctreated animals, the ADC lesion volume increased from 25 minutes to 45minutes post MCAO as in the placebo group. However, at the 70 minutetime point, just after initiation of Tat-NR2B9c drug, the increase wasattenuated. At 120 minutes and beyond, the ADC lesion inTat-NR2B9c-treated animals remained significantly smaller than inplacebo-treated rats. CBF-derived volumes and TTC infarct volumes weresignificantly smaller in Tat-NR2B9c animals. FIG. 5 indicates theabsolute mismatch between CBF and ADC derived lesion volumes. Relativeto placebo animals, the ADC/CBF mismatch lesion volumes weresignificantly larger starting at 90 minutes following inclusion in theTat-NR2B9c group. These results suggest that Tat-NR2B9c does not reduceinfarct size or improve outcome by shrinking the size of the ischemicpenumbra, as would occur if per-infarct blood flow were augmented by thetreatment. Rather, it suggests that Tat-NR2B9c works as aneuroprotectant that enhances the resilience of ischemic tissue toexisting ischemia.

Primary neuronal cultures treated with 100 nM Tat-NR2B9c demonstrated nosignificant in cell death or ATP levels compared to saline controls unnormoxic conditions. However, pretreated with 100 nM Tat-NR2B9c resultedin a 26% reduction in cell death as compared to saline controls 20 hoursafter OGD (FIG. 4A). ATP levels were 34% greater in Tat-NR2B9c treatedcultures 1 hour after OGD compared to saline treated cultures (FIG. 4B).

Discussion: Administration of Tat-NR2B9c 60 minutes after MCAO in ratsor mice resulted in a significant reduction in ADC hypointensity volumeand correlated with a reduction in infarct volume by TTC. Reduction instroke volume was associated with significant improvement inneurological scores 24 hours after MCAO. These anatomical andneurological improvements in stroke outcome after pMCAO were achievedwithout affecting cerebral blood flow, establishing that stroke therapyis achievable by neuroprotection even without blood flow augmentation.Thus, it is expected that coupling treatment with PSD-95 inhibitors toreperfusion strategies, either physical or therapeutic, are likely tofurther enhance salvage or neuronal tissues or the brain followingstroke.

Following onset of ischemia, there is a rapid drop in intracellular ATPlevels in neurons and glia that is associated with disruption ofhomeostatic mechanisms, failure of cellular function and cell death. AsPSD-95 inhibitor treatment resulted in decreased ATP depletion. Theseresults constitute the PSD-95 inhibitor treatment preserves the ischenicpenumbra providing a viable approach to extending the therapeutic ortemporal window of reperfusion therapies.

Example 3: A Single 4-5 Minute Infusion of Tat-NR2B9c is Sufficient toDisrupt the NMDAR:PSD95 Complex in Rodent Brains Subjected to Stroke

Methods Summary:

Postsynaptic Density-95 Inhibitors

Tat-NR2B9c is a synthetic peptide comprised of the 9 c-terminalamino-acids of the NR2B subunit (KLSSIESDV) fused to the cell membraneprotein transduction domain of the HIV-1-Tat protein (YGRKKRRQRRR; Tat).A control incapable of binding PSD95 is a similar peptide in which the 3terminal amino acids of the NR2B C-terminus sequence were switched fromSDV to ADA this control is termed “ADA” peptide. The peptides wereadministered intravenously in saline over 4 to 5 minutes by anindividual blinded to the identity of the compound and to its dose.

Animals

Adult male Sprague-Dawley rats (250 to 300 g; Charles RiverLaboratories, Sherbrooke, Quebec, Canada) were used according toprocedures approved by the institutional animal care committees. Allexperiments were performed on unfasted animals.

Surgical Preparation:

For permanent pial vessel occlusion (3 PVO), rats were anesthetized with100 mg/kg ketamine, 2 mg/kg acepromazine, and 50 mg/kg xylazine. Ratswere intubated and ventilated (60 strokes/min, tidal volume of 30 to 35mL). Mean arterial blood pressure, blood gases, pH, and glucose weremonitored with a left femoral artery catheter. Drug delivery was throughthe tail vein.

Experimental Procedure.

Animals were subjected to 3 PVO ischemia. One hour thereafter, they wereinjected with Saline, 3 nmol/g of Tat-KLSSIEADA (SEQ ID NO:76), or with0.3 nmol/g, 3 nmol/g and 10 nmol/g of Tat-NR2B9c (Tat-KLSSIESDV). Aftera further hour, the brain cortex was quickly harvested from theipsilateral (stroke) side, and from the side contralateral to thestroke. Sham treated animals had the craniotomy only, but no stroke ordrug infusion. Co-IP experiments were then conducted on the harvestedtissue using routine methods. Following CoIP with anti-NR2B oranti-PSD95 antibodies, the blots were probed with the indicatedantibodies (anti NR2B, PSD95 and Src). Densitometric analysis of bandswas performed using Image J software. To measure the effects of atreatment on the CoIP of associated proteins, the levels of each proteinon the blot were first normalized to the levels of the protein that wasimmunoprecipitated (PSD95 or NR2B), and then levels from the ipsilateral(stroke) side were normalized to the levels on the contralateral side.

Results:

The experiment was conducted in 5 replicates. Tissue for each replicatewas obtained from a 6 separate rats (one per condition). In eachreplicate, tissue was obtained from both the stroke side (ipsilateral)and the contralateral hemisphere. There was no effect of sham treatment(no stroke and no drug infusion) on the co-immunoprecipitation of PSD95and NR2B as evaluated by IPs performed with antibodies to either protein(FIGS. 9A-B shown as example). Similarly, the treatment with the controlpeptide Tat-KLSSIEADA had no effect on the co-IP of PSD95 and NR2B.However, treatment with Tat-KLSSIESDV inhibited the co-IP of PSD95 withNR2B in a dose-dependent manner (FIGS. 9A-B). The degree inhibition theco-IP of PSD95 with NR2B paralleled the potency of Tat-NR2B9c ininhibiting strokes, where 0.3 nmol/g is ineffective in reducing the sizeof the infarction in this model of stroke and doses of 3 or 10 nmol/gwere effective. Specifically, treatment with an Tat-NR2B9c dose of 0nM/g or 0.3 nM/g, which was ineffective in reducing stroke size, did notsignificantly inhibit the association of PSD95 and NR2B. By contrast,treatment with Tat-NR2B9c at a dose of 3 nM/g or 10 nM/g, which areeffective in inhibiting stroke damage also significantly inhibited theassociation of NR2B with PSD95. As a further control, tissue that wasimmunoprecipitated with either PSD95 or with NR2B was probed withantibodies against the NMDAR-associated protein kinase Src, a majorregulatory protein in the NMDAR signaling complex 25. Treatment with 10nM/g Tat-NR2B9c, which dissociates NR2B from PSD95 (FIG. 9A-B) had noeffect on the association of either PSD95 or of NR2B with Src (notshown). This indicates that the actions of Tat-NR2B9c in inhibitingNR2B/PSD95 interactions are specific, as Tat-NR2B9c had no effect on asimilar interaction of either protein with Src.

Conclusions:

First, Tat-NR2B9c gets into the brain on the side of the stroke and isable to dissociate pre-formed NR2B/PSD95 complexes in the ischemic brainwhen administered after a stroke. Second, Tat-NR2B9c is able to achievethis in a dose-dependent manner. Third, the doses at which Tat-NR2B9cachieves a significant dissociation of pre-formed NR2B/PSD95 complexesin the brain correspond to doses at which Tat-NR2B9c is neuroprotectivein the same animal stroke model. Fourth, the doses at which Tat-NR2B9cis unable to achieve a significant dissociation of pre-formed NR2B/PSD95complexes in the brain are also doses at which Tat-NR2B9c at whichTat-NR2B9c is not neuroprotective in the same animal stroke model Fifth,Tat-NR2B9c achieves its effects on NR2B/PSD95 complexes selectively(i.e., this is not a non-specific effect on NMDAR signaling complexmolecules).

A second study was performed with the same model and treatmentconditions to explore the time course of the disruption of thePSD95-NMDAR complex in neurons located in regions of the brain subjectedto a stroke. As with the previous study, rat brains were harvested at 2,4, 8, 24 or 48 hours after PIAL vessel occlusion to induce a stroke.FIG. 10 demonstrates that the NMDAR:PSD-95 complex is disrupted for atleast 8 hours following a single intravenous infusion of Tat-NR2B9c andpotentially 24-48 hours. Thus, there is a wide window of time betweenwhen Tat-NR2B9c could be administered to a patient and when the patientmay benefit from reperfusion therapy, whether therapeutic or mechanical.

Detailed Materials and Methods

Materials.

NA1 (GMP lot 165Il 107) and TAT-ADA (YGRKKRRQRRRKLSSIEADA) (SEQ ID NO:9)were chemically synthesized by Bachem and GeneScript, respectively. Allpeptides were high-performance liquid chromatography purified to >95%.Peptide stocks (3 mM or 10 mM) were prepared in sterile saline andstored at 4° C.

Antibodies.

Immunoprecipitations and westerns: Mouse anti-NMDA2B (ab28373; Abcam),rabbit anti-PSD95 (2507; Cell Signaling), rabbit anti-NMDA2B (4212, CellSignaling), mouse monoclonal anti-PSD95 (MA1-046, Thermo), mousemonoclonal anti-Src (ab16885-100) (Abcam). Dynabeads protein GImmunoprecipitation Kit was used for the immunoprecipitations (100.07D,Invitrogen). The secondary antibodies for western blots were peroxidaseconjugated AffiniPure F(ab′)2 Fragment Goat anti Rabbit IgG antibody(111-036-047) and peroxidase conjugated AffiniPure F(ab′)2 Fragment Goatanti Mouse IgG (115-036-006) from Jackson ImmunoResearch Lab Inc.

Three Pial Vessel Occlusion Model of Ischemia.

Male Sprague Dawley rats (n=6 per co-IP experiment) weighing between 250and 300 g were used for this study. For permanent three pial vesselocclusion (3 PVO) was performed as described previously 4,26. In brief,rats were anesthetized with a 0.5 ml/kg intramuscular injection ofketamine (100 mg/kg), acepromazine (2 mg/kg), and xylazine (5 mg/kg),supplemented with one-third of the initial dose as required. An analtemperature probe was inserted, and the animal was placed on a heatingpad maintained at 37° C. The skull was exposed via a midline incisionand scraped free of tissue. Using a dissecting microscope and apneumatic dental drill, a 6- to 8-mm cranial window was made over theright somatosensory cortex (2 mm caudal and 5 mm lateral to bregma) bydrilling a rectangle through the skull and lifting off the piece ofskull while keeping the dura intact. The 3 pial arteriolar middlecerebral artery branches around the barrel cortex (were electricallycauterized at 2 spots for per artery through the dura. After thecauterizations, the scalp was sutured. One hour after 3 PVO ischemia,rats were administered the treatment drug in a total of ˜300 ul ofsaline over 3 min through the femoral vein (exact volume to achieve thetarget mg/kg dose to the animal was determined by weight of the animal).At 1 hour after administration of the treatment drug (2 hours poststroke), rats were euthanized using 3% isoflurane mixed with oxygen.

Preparation of Brain Lysates.

Brains were removed from skull and both the ischemic area of the cortexand an equivalent sample on the contralateral side were quicklyharvested. Each brain sample was placed in 350 ul RIPA lysis buffer(Tris-HCL 50 mM, NaCl 150 mM, EDTA 1 mM, SDS 0.1%, Deoxycholic acid0.5%, NP-40 1% plus complete protease inhibitor cocktail (PhosSTOPphosphatase inhibitor cocktail, Roche)), homogenized and placed on ice.

Co-Immunoprecipitation of NMDAR and its Associated Proteins from RatCortex.

Rat cortex lysates were incubated on ice for one hour then centrifuged20 min at 4° C. (12,000 rpm). The supernatants were transferred to newtubes, incubated overnight at 4° C. with 30 ul Dynabeads protein G(Invitrogen) that were pre-loaded with 5 ug of either anti-PSD95 oranti-NMDAR antibodies (per Manufacturer's protocol using wash buffersprovided). Dynabead-antibody-antigen complexes were washed four times,and resuspended in 30 ul RIPA buffer+10 ul SDS-PAGE loading buffer (2.4ml 1M Tris pH 6.8, 0.8 g SDS, 4 ml 100% glycerol, 0.1% Bromophenol Blue,1 ml beta-mercaptoethanol, q.c. to 10 mL with dH2O). Samples were heatedfor 10 minutes at 75° C., placed on a magnet to retain the beads andthen supernatants were loaded onto an SDS-PAGE gel for analysis.

SDS-PAGE and Western Blotting.

Isolated immunoprecipitates were resolved using 10% SDS-PAGE andsubsequently transferred to nitrocellulose membranes. The membranes wereprobed with anti-PSD95 at 1:1000, then washed and developed using an ECLchemiluminescence kit (Amersham/GE Healthcare). Images were capturedusing a Luminescent Image Analyzer LAS-3000 (Fujifilm) with exposuresfrom 30 s to 2 min. Membranes were subsequently stripped for 10 minutesat room temperature (1.5% glycine, 0.1% SDS, 1% Tween 20 pH 2.2) andreblocked. Membranes were then re-probed with anti-NMDAR2B (1:1000) andanti-Src antibodies (1:500), washed, developed and images captured asabove.

Image Analysis.

Band intensities on images were analyzed by using Image J (NIH).Ipsilateral and contralateral bands were first normalized toimmunoprecipitated PSD95 or NR2B levels before generating ratios of banddensities between ipsilateral and contralateral immunoprecipitates fromthe same animal.

Example 4: Tat-NR2B9c and tPA can be Given Concurrently or at SeparateTimes to Improve Outcomes from Stroke

First, an in vitro study was performed to demonstrate that Tat-NR2B9chas no effect on clot lysis and that Tat-NR2B9c does not affect theability or rate of tPA to release fibrin from clots. Briefly, humanplasma containing ¹²⁵I labeled fibrinogen was incubated with variousconcentrations of Tat-NR2B9c, Tat-NR2B9c+1500 ng/ml tPA, or tPA alone ina buffer containing 100 nM NaCL, 30 nM Na₂HPO₄, 3 mM 30 nM NaH₂PO₄,pH7.4 for 2 hours at 37° C. Quantification of fibrin release from clotswas measured by scintillation counting of soluble material Tat-NR2B9cdid not lyse clots, nor did it affect the ability of tPA to releasefibrinogen from clots.

To demonstrate that Tat-NR2B9c and tPA could be given simultaneously inanimal models of stroke, we again used the PIAL occlusion model ofstroke in rats as above. Tat-NR2B9c was given as a 4-5 minuteintravenous infusion 1 hour after the stroke, and tPA was given asprescribed in humans (10% of the dose by weight as a bolus followed bythe remaining 90% of the dose given as an infusion over 1 hour) but withdose levels appropriate for rodent studies (10 times the human dose byweight). Groups (n=10) also included animals given both Tat-NR2B9c andtPA by these dose strategies concurrently, or with tPA initiated 15minutes after the infusion of Tat-NR2B9c. FIG. 11 demonstrates thatTat-NR2B9c plus tPA is more effective than tPA alone when given eitherconcurrently or when the Tat-NR2B9c dose precedes the tPA dose. Althoughthe efficacy of the combined treatment in rats is similar to that ofTat-NR2B9c alone, the efficacy of the combined treatments probablyreflects contributions of both tPA and Tat-NR2B9c because some of theTat-NR2B9c is subject to cleavage as the result of tPA convertingplasminogen to the protease plasmin, which is able to cleave Tat-NR2B9cin plasma. In other words, the data is consistent with activity lost asa result of Tat-NR2B9c being cleaved by plasmin being compensated for bytPA-mediated reperfusion. Cleavage of Tat-NR2B9c by plasmin is expectedto occur to a lesser extent in humans than rats because the dose of tPA(by weight) for activation of plasminogen is ten-fold less in humansthan rats. Thus, these data provide evidence that in humans thecontribution of tPA can combined with that of Tat-NR2B9c in reducingdamaging effects of stroke or other ischemia to the CNS with greatereffect than either agent alone.

Although the invention has been described in detail for purposes ofclarity of understanding, it will be obvious that certain modificationsmay be practiced within the scope of the appended claims. Allpublications, accession numbers, and patent documents cited in thisapplication are hereby incorporated by reference in their entirety forall purposes to the same extent as if each were so individually denoted.To the extent more than one sequence is associated with an accessionnumber at different times, the sequences associated with the accessionnumber as of the effective filing date of this application is meant. Theeffective filing date is the date of the earliest priority applicationdisclosing the accession number in question. Unless otherwise apparentfrom the context any element, embodiment, step, feature or aspect of theinvention can be performed in combination with any other.

1. A method of treating a damaging effect of ischemia on the centralnervous system, comprising administering a PSD-95 inhibitor to a subjecthaving or at risk of ischemia, and performing reperfusion therapy on thesubject, wherein the PSD-95 inhibitor and reperfusion therapy treat adamaging effect of the ischemia on the central nervous system of thesubject.
 2. The method of claim 1, wherein the PSD-95 inhibitor isadministered before reperfusion therapy is performed.
 3. The method ofclaim 1, wherein the PSD-95-inhibitor is administered to a subject atrisk of ischemia before onset of ischemia and the reperfusion therapy isperformed after onset of ischemia.
 4. The method of claim 1, wherein thePSD-95-inhibitor is administered and reperfusion therapy is performedafter onset of ischemia.
 5. The method of claim 1, wherein the ischemiais cerebral ischemia.
 6. The method of claim 1, wherein the subject hasa stroke.
 7. The method of claim 1, wherein the ischemia is cardiac,pulmonary or major limb ischemia affecting the central nervous system byinhibiting blood flow to or from the CNS.
 8. The method of claim 1,wherein the subject is tested for presence of cerebral ischemia and/orabsence of cerebral hemorrhage between administration of the PSD-95inhibitor and performance of the reperfusion therapy. 9-11. (canceled)12. The method of claim 1, wherein the PSD-95-inhibitor is tat-NR2B9c.13. The method of claim 1, wherein the reperfusion is performed byadministering a thrombolytic agent.
 14. The method of claim 13, whereinthe thrombolytic agent is a plasminogen activator.
 15. The method ofclaim 14, wherein the thrombolytic agent is tPA.
 16. The method of claim13, wherein the interval between administering PSD-95 and reperfusiontherapy is 30 min to 6 hr.
 17. The method of claim 13, wherein thethrombolytic agent is administered by localized administration to a siteof impaired blood flow.
 18. The method of claim 1, wherein thereperfusion therapy is mechanical reperfusion.
 19. The method of claim1, wherein the reperfusion therapy is performed more than 3 hours afteronset of ischemia.
 20. The method of claim 1, wherein the reperfusiontherapy is performed more than 4.5 hours after onset of ischemia. 21.The method of claim 1, wherein the reperfusion therapy is performed morethan 4.5 hours and less than 24 hours after onset of ischemia.
 22. Themethod of claim 1, wherein the reperfusion therapy is performed afterdetermining the subject qualifies for reperfusion based on lack of acompleted infarction, an ischemic penumbra and lack of hemorrhage asshown by CT, MRI or PET analysis.
 23. The method of claim 22, whereinthe reperfusion therapy is performed at least 12 or at least 24 hoursafter onset of ischemia.
 24. The method of claim 1, wherein thereperfusion therapy is performed 275-690 minutes after onset ofischemia.
 25. The method of claim 11, wherein the peptide is linked toan internalization peptide or is lipidated thereby facilitating passageof the peptide across a cell membrane or the blood brain barrier. 26.(canceled)
 27. The method of claim 26, wherein peptide has an amino acidsequence consisting or comprising of KLSSIESDV (SEQ ID NO:5) orKLSSIETDV (SEQ ID NO:43).
 28. (canceled)
 29. A method of treating asubject population presenting sign(s) and/or symptom(s) of ischemia,comprising administering a PSD-95 inhibitor to the subjects; wherein thesubjects are analyzed for unacceptable risk of side effects ofreperfusion therapy, and subjects without unacceptable risk of sideeffects receive reperfusion therapy and subjects with unacceptable riskof side effects do not receive reperfusion therapy. 30-35. (canceled)